EP0901527A2 - Regulating metabolism by modifying the level of trehalose-6-phosphate - Google Patents

Abstract

The invention lies in the field of regulation of carbon flow in the metabolism of the cell. It has been found that induction of a change in the intracellular availability of the saccharide trehalose-6-phosphate (T-6-P) induces modifications of the development and/or composition of cells, tissue and organs in vivo. These changes can be induced by introducing or inhibiting the enzymes trehalose phosphate synthase (TPS) which is capable of forming T-6-P and trehalose phosphate phosphatase (TPP) which degrades T-6-P into trehalose. The carbon flow through the glycolysis will be stimulated by a decrease in the intracellular level of T-6-P.

Description

REGULATING METABOLISM BY MODIFYING THE LEVEL OF TREHALOSE-6-PHOSPHATE

FIELD OF THE INVENTION

Glycolysis has been one of the first metabolic processes described in biochemical detail in the literature Although the general flow of carbohydrates in organisms is known and although all enzymes of the glycolytic pathway(s) are elucidated, the signal which determines the induction of metabolism by stimulating glycolysis has not been unravelled Several hypotheses, especially based on the situation in yeast have been put forward, but none has been proven beyond doubt.

Influence on the direction of the carbohydrate partitioning does not only influence directly the cellular processes of glycolysis and carbohydrate storage, but it can also be used to influence secondary or derived processes such as cell division, biomass generation and accumulation of storage compounds, thereby determining growth and productivity Especially in plants, often the properties of a tissue are directly influenced by the presence of carbohydrates, and the steering of carbohydrate partitioning can give substantial differences

The growth, development and yield of plants depends on the energy which such plants can derive from Cθ₂-fιxatιon during photosynthesis

Photosynthesis primarily takes place in leaves and to a lesser extent m the stem, while other plant organs such as roots, seeds or tubers do not essentially contribute to the photoassimilation process These tissues are completely dependent on photosynthetically active organs for their growth and nutrition. This then means that there is a flux of products derived from photosynthesis (collectively called "photosynthate") to photosynthetically inactive parts of the plants

The photosynthetically active parts are denominated as "sources" and they are defined as net exporters of photosynthate The photosynthetically inactive parts are denominated as "sinks" and they are defined as net importers of photosynthate

It is assumed that both the efficiency of photosynthesis, as well as the carbohydrate partitioning in a plant are essential Newly developmg tissues like young leaves or other parts like root and seed are completely dependent on photosynthesis m the sources The possibility of influencing the carbohydrate partitioning would have great impact on the phenotype of a plant, e g . its height, the mternodium distance, the size and form of a leaf and the size and structure of the root system.

Furthermore, the distribution of the photoassimilation products is of great importance for the yield of plant biomass and products. An example is the development in wheat over the last century. Its photosynthetic capacity has not changed considerably but the yield of wheat grain has increased substantially, i . e the harvest index (ratio harvestable biomass/total biomass) has increased. The underlying reason is that the sink-to-source ratio was changed by conventional breeding, such that the harvestable sinks, i . e seeds, portion increased. However, the mechanism which regulates the distribution of assimilation products and consequently the formation of sinks and sources is yet unknown. The mechanism is believed to be located somewhere m the carbohydrate metabolic pathways and their regulation. In the recent research it has become apparent that hexokmases may play a major role in metabolite signalling and control of metabolic flow. A number of mechanisms for the regulation of the hexokinase activity have been postulated (Graham et al. (1994), The Plant Cell 6. 761; Jang & Sheen (1994), The Plant Cell 6, 1665, Rose et al . Eur. J. Biochem. 199, 511-518, 1991, Blazquez et al (1993), FEBS 329, 51; Koch, Annu. Rev Plant Physiol Plant. Mol. Biol. (1996) 47, 509; Jang et al. (1997), The Plant Cell 9, 5. One of these theories of hexokinase regulation, postulated m yeast mentions trehalose and its related monosaccharides (Thevelem & Hohmann (1995), TIBS 20, 3) However, it is hard to see that this would be an universal mechanism, as trehalose synthesis is believed to be restricted to certain species.

Thus, there still remains a need for the elucidation of the signal which can direct the modification of the development and/or composition of cells, tissue and organs in vivo . SUMMARY OF THE INVENTION

It has now been found that modification of the development and/or composition of cells, tissue and organs m vivo is possible by introducing the enzyme trehalose-6-phosphate synthase (TPS) and/or trehalose-6-phosphatase phosphate (TPP) thereby inducing a change m metabolic pathways of the saccharide trehalose-6-phosphate (T-6-P) resulting m an alteration of the intracellular availability of T-6-P. Introduction of TPS thereby inducing an increase in the intracellular concentration of T-6-P causes inhibition of carbon flow m the glycolytic direction, stimulation of the photosynthesis, inhibition of growth, stimulation of sink-related activity and an increase in storage of resources. Introduction of TPP thereby introducing a decrease in the intracellular concentration of T-6-P causes stimulation of carbon flow in the glycolytic direction, increase m biomass and a decrease in photosynthetic activity.

The levels of T-6-P may be influenced by genetic engineering of an organism with gene constructs able to influence the level of T-6-P or by exogenously (orally, topically, parenterally etc ) supplying compounds able to influence these levels.

The gene constructs that can be used in this invention are constructs harbouring the gene for trehalose phosphate synthase (TPS) the enzyme that is able to catalyze the reaction from glucose-6-phosphate and UDP-glucose to T-6-P. On the other side a construct coding for the enzyme trehalose-phosphate phosphatase (TPP) which catalyzes the reaction from T-6-P to trehalose will, upon expression, give a decrease of the amount of T-6-P

Alternatively, gene constructs harbouring antisense TPS or TPP can be used to regulate the intracellular availability of T-6-P. Furthermore, it was recently reported that an intracellular phospho-alpha- (1, 1)-glucosidase, TreA, from Baci ll us subtil is was able to hydrolyse T-6-P into glucose and glucose-6-phosphate (Schock et al., Gene, 170. 77-80, 1996) . A similar enzyme has already been described for E. col i (Rimmele and Boos (1996), J. Bact. 176 (18), 5654-) .

For overexpression heterologous or homologous gene constructs have to be used. It is believed that the endogenous T-6-P forming and/or degrading enzymes are under allosteric regulation and regulation through covalent modification This regulation may be circumvented by using heterologous genes.

Alternatively, mutation of heterologous or homologous genes may be used to abolish regulation. The invention also gives the ability to modify source-sink relations and resource allocation in plants. The whole carbon economy of the plant, including assimilate production in source tissues and utilization in source tissues can be modified, which may lead to increased biomass yield of harvested products. Using this approach, increased yield potential can be realized, as well as improved harvest index and product quality These changes in source tissues can lead to changes in sink tissues by for instance increased export of photosynthase Conversely changes in sink tissue can lead to change in source tissue. Specific expression m a cell organelle, a tissue or other part of an organism enables the general effects that have been mentioned above to be directed to specific local applications. This specific expression can be established by placing the genes coding for TPS, TPP or the antisense genes for TPS or TPP under control of a specific promoter.

Specific expression also enables the simultaneous expression of both TPS and TPP enzymes in different tissues thereby increasing the level of T-6-P and decreasing the level of T-6-P locally.

By using specific promoters it is also possible to construct a temporal difference For this purpose promoters can be used that are specifically active during a certain period of the organogenesis of the plant parts. In this way it is possible to first influence the amount of organs which will be developed and then enable these organs to be filled with storage material like starch, oil or proteins. Alternatively, inducible promoters may be used to selectively switch on or off the expression of the genes of the invention, induction can be achieved by for instance pathogens, stress, chemicals or light/dark stimuli. DEFINITIONS

Hexokinase activity is the enzymatic activity found in cells which catalyzes the reaction of hexose to hexose-6-phosphate. Hexoses include glucose, fructose, galactose or any other Cg sugar. It is acknowledged that there are many isoenzymes which all can play a part in said biochemical reaction. By catalyzing this reaction hexokinase forms a key enzyme in hexose (glucose) signalling. - Hexose signalling is the regulatory mechanism by which a cell senses the availability of hexose (glucose) .

Glycolysis is the sequence of reactions that converts glucose into pyruvate with the concomitant production of ATP. Cold sweetening is the accumulation of soluble sugars m potato tubers after harvest when stored at low temperatures.

Storage of resource material is the process in which the primary product glucose is metabolized into the molecular form which is fit for storage in the cell or in a specialized tissue. These forms can be divers. In the plant kingdom storage mostly takes place in the form of carbohydrates and polycarbohydrates such as starch, fructan and cellulose, or as the more simple mono- and di-saccharides like fructose, sucrose and maltose; m the form of oils such as arachic or oleic oil and m the form of proteins such as crucifeπn, napm and seed storage proteins m rapeseed. In animal cells also polymeric carbohydrates such as glycogen are formed, but also a large amount of energy rich carbon compounds is transferred into fat and lipids. Biomass is the total mass of biological material .

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DESCRIPTION OF THE FIGURES

Figure 1. Schematic representation of plasmid pVDH275 harbouring the neomycm-phosphotransferase gene (NPTII) flanked by the 35S cauliflower mosaic virus promoter (P35S) and terminator (T35S) as a selectable marker; an expression cassette comprising the pea plastocyanin promoter (pPCpea) and the nopaline synthase terminator (Tnos) , right (RB) and left (LB) T-DNA border sequences and a bacterial kanamycin resistance (KanR) marker gene.

Figure 2. Northern blot analysis of transgenic tobacco plants. Panel A depicts expression of otsA mRNA in leaves of individual pMOG799 transgenic tobacco plants. The control lane "C" contains total RNA from a non-transformed N. tabacum plant.

Figure 3 Lineup of plant derived TPS encoding sequences compared with the TPS_yeast sequence using the Wisconsin GCG sequence analysis package (Devereux et al (1984) A comprehensive set of sequence analysis programs of the VAX Nucl. Acids Res., 12, 387) TPSatal 3/56 and 142 TPSrιce3 (SEQ ID NO: 53) and RiceTPS code for respectively Arabidopsis and Rice TPS enzymes derived from EST database sequences.

TPSsunlO, TPSsel43, (SEQ ID NO.44) and TPSselθ (SEQ ID NO 42) code for respectively sunflower and Selagmella TPS enzymes derived from sequences isolated by PCR techniques (see example 3)

Figure 4. Alignment of PCR amplified tobacco TPS cDNA fragments with the TPS encoding yeast TPS1 gene. Boxes indicate identity between ammo-acids of all four listed sequences.

Figure 5. Alignment of PCR amplified tobacco TPP cDNA fragments with the TPP encoding yeast TPS2 gene. Boxes indicate identity between ammo-acids of all four listed sequences

Figure 6. Alignment of a fragment of the PCR amplified sunflower TPS/TPP bipartite cDNA (SEQ ID NO: 24) with the TPP encoding yeast TPS2 gene. Boxes indicate identity between ammo-acids of both sequences. ?

Figure 7 Alignment of a fragment of the Arabidopsis TPSl and Rice EST clones with the TPS encoding yeast TPSl gene. Boxes indicate identity between ammo-acids of all three sequences.

Figure 8. Alignment of a fragment of the PCR amplified human TPS cDNA (SEQ ID NO: 10) with the TPS encoding yeast TPSl gene. Boxes indicate identity between ammo-acids of both sequences.

Figure 9 Trehalose accumulation in tubers of pMOG1027 (35S as- trehalase) transgenic potato plants

Figure 10 Hexokinase activity of a wild-type potato tuber (Solanum tuberoεum cv. Kardal) extract with and without the addition of trehalose-6-phosphate.

Figure 11. Hexokinase activity of a wild-type potato tuber ( Solanum tuberosum cv. Kardal) extract with and without the addition of trehalose-6-phosphate. Fructose or glucose is used as substrate for the assay

Figure 12 Hexokinase activity of a wild-type tobacco leaf extract [Nicotiana tabacum cv. SRI) with and without the addition of trehalose-6-phosphate Fructose or glucose is used as substrate for the assay.

Figure 13. Plot of a tobacco hexokinase activity measurement. Data series 1 : Tobacco plant extract

Data series 2. Tobacco plant extract + 1 mM trehalose-6-phosphate Data series 3: Commercial hexokinase extract from yeast (1/8 unit)

Figure 14. Hexokinase activity of a wild-type rice leaf extract { Oryza sa ti va ) extract with and without the addition of trehalose-6- phosphate. Experiments have been performed in duplicate using different amounts of extracts. Fructose or glucose is used as substrate for the assay. Figure 15. Hexokinase activity of a wild-type maize leaf extract ( Zea mais) extract with and without the addition of trehalose-6-phosphate. Fructose or glucose is used as substrate for the assay.

Figure 16. Fluorescence characteristics of wild-type (triangle), PC- TPS (square) and 35S-TPP (cross) tobacco leaves. The upper two panels show the electron transport efficiency (ETE) at the indicated light intensities (PAR) . Plants were measured after a dark-period (upper- left panel) and after a light-period (upper-right panel) . The bottom panels show reduction of fluorescence due to assimilate accumulation (non-photochemical quenching) . Left and right panel as above.

Figure 17 Relative sink-activity of plant-parts of PC-TPS (Famine) and 35S-TPP (Feast) transgenic tobacco plants. Indicated is the nett C-accumulation expressed as percentage of total C-content, for various plant-parts after a period of light (D) or light + dark (D + N) .

Figure 18. Actual distribution of carbon in plant-parts of PC-TPS (Famine) and 35S-TPP (Feast) transgenic tobacco plants. Indicated is the nett C-accumulation expressed as percentage of total daily accumulated new C for various plant-parts after a period of light (D) or light + dark (D + N) .

Figure 19. Reduced and enhanced bolting m transgenic lettuce lines expressing PC-TPS or PC-TPP compared to wild-type plants. The lower panel shows leaf morphology and colour.

Figure 20. Profile of soluble sugars (Fig. 20/1) in extracts of transgenic lettuce (upper panel) and transgenic beet (lower panel) lines In the upper panel controls are GUS-transgemc lines which are compared to lines transgemcs for PC-TPS and PC-TPP. In the lower panel all transgenic are PC-TPS. Starch profiles are depicted in Fig. 20/2. Figure 21. Plant and leaf morphology of transgenic sugarbeet lines expressing PC-TPS (TPS) or PC-TPP (TPP) compared to wild-type plants (Control) . TPS A-type has leaves which are comparable to wild-type while TPS D-type has clearly smaller leaves. The leaves of the TPP transgenic line have a lighter green colour, a larger petiole and an increased size compared to the control.

Figure 22. Taproot diameter of transgenic sugarbeet lines (PC-TPS) . In the upper panel A, B, C and D indicate decreasing leaf sizes as compared to control (A) . In the lower panel individual clones of control and PC-TPS line 286-2 are shown.

Figure 23. Tuber yield of pMOG799 (35S TPS) transgenic potato lines.

Figure 24. Tuber yield of pMOGlOlO (35Ξ TPP) and pMOG1124 (PC-TPP) transgenic potato lines.

Figure 25 Tuber yield of 22 independent wild-type S. tuberosum clones.

Figure 26. Tuber yield of pMOG1093 (PC-TPS) transgenic potato lines in comparison to wild-type. B, C, D, E, F, G indicate decreasing leaf sizes as compared to wild-type (B/C) .

Figure 27. Tuber yield of pMOG845 (Pat-TPS) transgenic potato lines (Figure 27-1) in comparison to wild-type (Figure 27-2). B, C indicate leaf sizes.

Figure 28. Tuber yield of pMOG1129 (845-11/22/28) transgenic potato lines.

Figure 29. Cross section through leaves of TPP (lower panel) and TPS (upper panel) transgenic tobacco plants. Additional cell layers and increased cell size are visible in the TPS cross section. I O

Figure 30. HPLC-PED analysis of tubers transgenic for TPS_{E coll} before and after storage at 4°C. Kardal C, F, B, G and H are non-transgenic control lines .

Figure 31. Leaf morphology, colour and size of tobacco lines transgenic for 35S TPS (upper leaf), wild-type (middle leaf) and transgenic for 35S TPP (bottom leaf) .

Figure 32. Metabolic profiling of 35S TPS (pMOG799), 35S TPP (pMOGlOlO), wild-type (WT) , PC-TPS (pM0G1177) and PC-TPP (pM0G1124) transgenic tobacco lines. Shown are the levels of trehalose, soluble sugars (Figure 32-1), starch and chlorophyll (Figure 32-2)

Figure 33. Tuber yield of pMOG1027 (35S as-trehalase) and pMOG1027 (845-11/22/28) (35S as-trehalaεe pat TPS) transgenic potato lines in comparison to wild-type potato lines.

Figure 34. Starcn content of pMOG1027 (35S as-trehalaεe) and PMOG1027 (845-11/22/28) (35S as-trehalaεe pat TPS) transgenic potato lines in comparison to wild-type potato lines The sequence of all lines depicted is identical to Fig. 33.

Figure 35. Yield of pMOG1028 (pat as-trehalase) and pMOG1028 (845- 11/22/28) (pat as-trehalase pat TPS) transgenic potato lines m comparison to wild-type potato lines

Figure 36. Yield of pMOG1092 (PC as-trehalase) transgenic potato lines in comparison to wild-type potato lines as depicted m Fig 35.

Figure 37. Yield of pMOG1130 (PC as-trehalase PC TPS) transgenic potato lines m comparison to wild-type potato lines as depicted in Fig. 35. ; /

DETAILED DESCRIPTION OF THE INVENTION

The invention is concerned with the finding that metabolism can be modified m vivo by the level of T-6-P. A decrease of the intracellular concentration of T-6-P stimulates glycolytic activity On the contrary, an increase of the T-6-P concentration will inhibit glycolytic activity and stimulate photosynthesis.

These modifications established by changes m T-6-P levels are most likely a result of the signalling function of hexokinase, which activity is shown to be regulated by T-6-P. An increase in the flux through hexokinase (i.e. an increase in the amount of glucose) that is reacted m glucose-6-phosphate has been shown to inhibit photosynthetic activity in plants. Furthermore, an increase in the flux through hexokinase would not only stimulate the glycolysis, but also cell division activity

THEORY OF TREHALOSE-6-PHOSPHATE REGULATION OF CARBON

METABOLISM In a normal plant cell formation of carbohydrates takes place m the process of photosynthesis in which CO₂ is fixed and reduced to phosphorylated hexoses with sucrose as an end-product Normally this sucrose is transported out of the cell to cells or tissues which through uptake of this sucrose can use the carbohydrates as building material for their metabolism or are able to store the carbohydrates as e . g . starch In this respect, in plants, cells that are able to photosynthesize and thus to produce carbohydrates are denominated as sources, while cells which consume or store the carbohydrates are called sinks .

In animal and most microbial cells no photosynthesis takes place and the carbohydrates have to be obtained from external sources, either by direct uptake from saccharides (e g yeasts and other micro¬ organisms) or by digestion of carbohydrates (animals). Carbohydrate transport usually takes place in these organisms m the form of glucose, which is actively transported over the cell membrane

After entrance mto the cell, one of the first steps in the metabolic pathway is the phosphorylation of glucose mto glucose-6- phosphate catalyzed by the enzyme hexokinase It has been demonstrated that in plants sugars which are phosphorylated by hexokinase (HXK) are controlling the expression of genes involved in photosynthesis (Jang & I U.

Sheen (1994), The Plant Cell 6, 1665). Therefor, it has been proposed that HXK may have a dual function and may act as a key sensor and signal transmitter of carbohydrate-mediated regulation of gene- expression. It is believed that this regulation normally signals the cell about the availability of startmg product, i . e . glucose. Similar effects are observed by the introduction of TPS or TPP which influence the level of T-6-P. Moreover, it is shown that m vi tro T-6-P levels affect hexokinase activity. By increasing the level of T-6-P, the cell perceives a signal that there is a shortage of carbohydrate input. Conversely, a decrease m the level of T-6-P results in a signal that there is plenty of glucose, resulting in the down-regulation of photosynthesis: it signals that substrate for glycolysis and consequently energy supply for processes as cell growth and cell division is sufficiently available. This signalling is thought to be initiated by the increased flux through hexokinase (J J. Van Oosten, public lecture at RijksUmversiteit Utrecht dated April 19, 1996).

The theory that hexokinase signalling in plants can be regulated through modulation of the level of trehalose-6-phosphate would imply that all plants require the presence of an enzyme system able to generate and break-down the signal molecule trehalose-6-phosphate. Although trehalose is commonly found m a wide variety of fungi, bacterial, yeasts and algae, as well as m some invertebrates, only a very limited range of vascular plants have been proposed to be able to synthesize this sugar (Elbem (1974), Adv Carboh Chem Biochem. 30, 227) . A phenomenon which was not understood until now is that despite the apparent lack of trehalose synthesizing enzymes, all plants do seem to contain trehalases, enzymes which are able to break down trehalose mto two glucose molecules.

Indirect evidence for the presence of a metabolic pathway for trehalose is obtained by experiments presented herein with trehalase inhibitors such as Validamycm A or transformation with anti-sense trehalase.

Production of trehalose would be hampered if its intermediate T- 6-P would influence metabolic activity too much. Preferably, m order to accumulate high levels of trehalose without affecting partitioning and allocation of metabolites by the action of trehalose-6-phosphate, one should overexpress a bipartite TPS/TPP enzyme. Such an enzyme would resemble a genetic constitution as found m yeast, where the /3

TPS2 gene product harbours a TPS and TPP homologous region when compared with the E. col i otsA and otsB gene (Kaasen et al . (1994), Gene 145, 9) . Using such an enzyme, trehalose-6-phosphate will not become freely available to other cell components Another example of such a bipartite enzyme is given by Zentella & Iturriaga (Plant Physiol (1996), 111 Abstract 88) who isolated a 3 2 kb cDNA from Selagmella l epidophylla encoding a putative trehalose-6-phosphate synthase/phosphatase. It is also envisaged that construction of a truncated TPS-TPP gene product, whereby only the TPS activity would be retained, would be as powerful for synthesis of T-6-P as the otsA gene of E. coli , also when used in homologous systems

On a molecular level we have data that indicate that next to Selagmel la also trehalose synthesizing genes are present in Arabidopsis , tobacco, rice and sunflower. Using degenerated primers, based on conserved sequences between TPS_{E coll} and TPS_yeast, we have been able to identify genes encoding putative trehalose-6-phosphate generating enzymes m sunflower and tobacco. Sequence comparison revealed significant homology between these sequences, the TPS genes from yeast and E. coli , and EST (expressed sequences tags) sequences from Arabi dopsi s and rice (see also Table 6b which contains the EST numbers of homologous EST's found) .

Recently an Arabidopsis gene has been elucidated (disclosed m GENBANK Ace. No. Y08568, depicted m SEQ ID NO: 39) that on basis of its homology can be considered as a bipartite enzyme These data indicate that, m contrast to current beliefs, most plants do contain genes which encode trehalose-phosphate-synthases enabling them to synthesize T-6-P As proven by the accumulation of trehalose in TPS expressing plants, plants also contain phosphatases, non¬ specific or specific, able to dephosphorylate the T-6-P into trehalose. The presence of trehalase in all plants may be to effectuate turnover of trehalose.

Furthermore, we also provide data that T-6-P is involved m regulating carbohydrate pathways m human tissue. We have elucidated a human TPS gene (depicted in SEQ ID NO: 10) which shows homology with the TPS genes of yeast, E. coli and plants. Furthermore, we show data that also the activity of hexokinase is influenced in mammalian (mouse) tissue. M

Generation of the "plenty" signal by decreasing the intracellular concentration of trehalose-6-phosphate through expression of the enzyme TPP (or inhibition of the enzyme TPS) will signal all cell systems to increase glycolytic carbon flow and inhibit photosynthesis. This is nicely shown in the experimental part, where for instance m Experiment 2 transgenic tobacco plants are described in which the enzyme TPP is expressed having increased leaf size, increased branching and a reduction of the amount of chlorophyll. However, since the "plenty" signal is generated m the absence of sufficient supply of glucose, the pool of carbohydrates in the cell is rapidly depleted.

Thus, assuming that the artificial "plenty" signal holds on, the reduction m carbohydrates will finally become limiting for growth and cell division, i . e . the cells will use up all their storage carbohydrates and will be in a "hunger"-stage Thus, leaves are formed with a low amount of stored carbohydrates On the other hand, plants that express a construct with a gene coding for TPS, which increases the intracellular amount of T-6-P, showed a reduction of leaf size, while also the leaves were darker green and contained an increased amount of chlorophyll

In yeast, a manor role of glucose-induced signalling is to switch metabolism from a neogenetic/respirative mode to a fermentative mode Several signalling pathways are involved m this phenomenon (Thevelein and Hohmann, (1995) TIBS 20, 3) Besides the possible role of hexokinase signalling, the RAS-cyclic-AMP (cAMP) pathway has been shown to be activated by glucose. Activation of the RAS-cAMP pathway by glucose requires glucose phosphorylation, but no further glucose metabolism. So far, this pathway has been shown to activate trehalase and 6-phosphofructo-2-kmaεe (thereby stimulating glycolysis) , while fructose-1, 6-bιεphosphatase is inhibited (thereby preventing gluconeogenesis) , by cAMP-dependent protein phosphorylation. This signal transduction route and the metabolic effects it can bring about can thus be envisaged as one that acts in parallels with the hexokinase signalling pathway, that is shown to be influenced by the level of trehalose-6-phosphate

As described m our invention, transgenic plants expressing as- trehalase reveal similar phenomena, like dark-green leaves, enhanced / 5 yield, as observed when expressing a TPS gene. It also seems that expression of as-trehalase in double-constructs enhances the effects that are caused by the expression of TPS Trehalase activity has been shown to be present in e.g. plants, insects, animals, fungi and bacteria while only in a limited number of species, trehalose is accumulated.

Up to now, the role of trehalase m plants is unknown although this enzyme is present m almost all plant-species. It has been proposed to be involved in plant pathogen interactions and/or plant defense responses. We have isolated a potato trehalase gene and show that inhibition of trehalase activity in potato leaf and tuber tissues leads to an increase in tuber-yield. Fruit-specific expression of as- trehalase in tomato combined with TPS expression dramatically alters fruit development.

Accordmg to one embodiment of the invention, accumulation of T- 6-P is brought about in cells m which the capacity of producing T-6-P has been introduced by introduction of an expressible gene construct encoding trehalose-phosphate-synthase (TPS) . Any trehalose phosphate synthase gene under the control of regulatory elements necessary for expression of DNA in cells, either specifically or constitutively, may be used, as long as it is capable of producing a trehalose phosphate synthase capable of T-6-P production in said cells. One example of an open reading frame according to the invention is one encoding a TPS- enzyme as represented in SEQ ID NO: 2. Other examples are the open reading frames as represented m SEQ ID NO's: 10, 18-23, 41 and 45-53. As is illustrated by the above-mentioned sequences it is well known that more than one DNA sequence may encode an identical enzyme, which fact is caused by the degeneracy of the genetic code. If desired, the open reading frame encoding the trehalose phosphate synthase activity may be adapted to codon usage in the host of choice, but this is not a requirement.

The isolated nucleic acid sequence represented by for instance SEQ ID NO 2, may be used to identify trehalose phosphate synthase genes in other organisms and subsequently isolating and cloning them, by PCR techniques and/or by hybridizing DNA from other sources with a DNA- or RNA fragment obtainable from the E. col i gene. Preferably, such DNA sequences are screened by hybridizing under more or less stringent conditions (influenced by factors such as temperature and ionic strength of the hybridization mixture) . Whether or not conditions are stringent also depends on the nature of the hybridization, i . e . DNA:DNA, DNA:RNA, RNA-RNA, as well as the length of the shortest hybridizing fragment. Those of skill in the art are readily capable of establishing a hybridization regime stringent enough to isolate TPS genes, while avoiding non-specific hybridization. As genes involved in trehalose synthesis from other sources become available these can be used m a similar way to obtain an expressible trehalose phosphate synthase gene accordmg to the invention. More detail is given in the experimental section.

Sources for isolating trehalose phosphate synthase activities include microorganisms (e.g bacteria, yeast, fungi), plants, animals, and the like. Isolated DNA sequenceε encoding trehalose phosphate synthase activity from other sources may be uεed likewise m a method for producing T-6-P according to the invention As an example, genes for producing T-6-P from yeast are disclosed in WO 93/17093.

The invention also encompasses nucleic acid sequences which have been obtained by modifying the nucleic acid sequence represented in SEQ ID NO: 1 by mutating one or more codons so that it results in ammo acid changes in the encoded protein, as long as mutation of the ammo acid sequence does not entirely abolish trehalose phosphate synthase activity.

According to another embodiment of the invention the trehalose- 6-phosphate in a cell can be converted into trehalose by trehalose phosphate phosphatase encoding genes under control of regulatory elements necessary for the expression of DNA in cells. A preferred open reading frame according to the invention is one encoding a TPP- enzyme as represented in SEQ ID NO: 4 (Kaasen et al . (1994) Gene, 145, 9). It is well known that more than one DNA sequence may encode an identical enzyme, which fact is caused by the degeneracy of the genetic code. If desired, the open reading frame encoding the trehalose phosphate phosphatase activity may be adapted to codon usage in the host of choice, but this is not a requirement. The isolated nucleic acid sequence represented by SEQ ID NO- 3, may be used to identify trehalose phosphate phosphatase genes in other organisms and subsequently isolating and cloning them, by PCR techniques and/or by hybridizing DNA from other sources with a DNA- or 7

RNA fragment obtainable from the E. coli gene. Preferably, such DNA sequences are screened by hybridizing under more or less stringent conditions (influenced by factors such as temperature and ionic strength of the hybridization mixture) . Whether or not conditions are stringent also depends on the nature of the hybridization, l e . DNA:DNA, DNA:RNA, RNA:RNA, as well as the length of the shortest hybridizing fragment Those of skill in the art are readily capable of establishing a hybridization regime stringent enough to isolate TPP genes, while avoiding aspecific hybridization. As genes involved in trehalose synthesis from other sources become available these can be used in a similar way to obtain an expressible trehalose phosphate phosphatase gene according to the invention. More detail is given in the experimental section.

Sources for isolating trehalose phosphate phosphatase activities include microorganisms ( e . g bacteria, yeast, fungi), plants, animals, and the like Isolated DNA sequences encoding trehalose phosphate phosphatase activity from other sources may be used likewise

The mvention also encompasses nucleic acid sequences which have been obtained by modifying the nucleic acid sequence represented m SEQ ID NO. 3 by mutating one or more codons so that it results in ammo acid changes in the encoded protein, as long as mutation of the ammo acid sequence does not entirely abolish trehalose phosphate phosphatase activity. Other enzymes with TPS or TPP activity are represented by the so- called bipartite enzymes. It is envisaged that the part of the sequence which is specifically coding for one of the two activities can be separated from the part of the bipartite enzyme coding for the other activity. One way to separate the activities is to insert a mutation in the sequence coding for the activity that is not selected, by which mutation the expressed protein is impaired or deficient of this activity and thus only performs the other function. This can be done both for the TPS- and TPP-activity coding sequence. Thus, the coding sequences obtained m such a way can be used for the formation of novel chimaeric open reading frames capable of expression of enzymes having either TPS or TPP activity.

According to another embodiment of the invention, especially plants can be genetically altered to produce and accumulate the above- mentioned enzymes in specific parts of the plant. Preferred sites of 19 enzyme expression are leaves and storage parts of plants In particular potato tubers are considered to be suitable plant parts A preferred promoter to achieve selective TPS-enzyme expression in microtubers and tubers of potato is obtainable from the region upstream of the open reading frame of the patatm gene of potato. Another suitable promoter for specific expression is the plastocyanm promoter, which is specific for photoassirπilatmg parts of plants Furthermore, it is envisaged that specific expression in plant parts can yield a favourable effect for plant growth and reproduction or for economic use of said plants Promoters which are useful m this respect are the E8-promoter (EP 0 409 629) and the 2All-promoter (van Haaren and Houck (1993), Plant Mol Biol., 221, 625) which are fruit-specific, the cruciferm promoter the napm promoter and the ACP promoter which are seed-specific, the PAL- promoter, the chalcon-isomerase promoter which is flower-specific the SSU promoter, and ferredoxm promoter, which are leaf-specific the TobRb7 promoter which is root-specific, the RolC promoter which is specific for phloem and the HMG2 promoter (Enjuto et al (1995), Plant Cell 7, 517) and the rice PCNA promoter (Kosugi et al (1995), Plant J 7, 877) which are specific for meristematic tissue

Another option under this invention is to use inducible promoters Promoters are known which are inducible by pathogens, by stress, by chemical or light/dark stimuli It is envisaged that for induction of specific phenoma, for instance sprouting, bolting, seed setting, filling of storage tissues, it is beneficial to induce the activity of the genes of the invention by external stimuli This enables normal development of the plant and the advantages of the mducibility of the desired phenomena at control Promoters which qualify for use in such a regime are the pathogen inducible promoters described in DE 4446342 (fungus and auxin inducible PRP-1), WO

96/28561 (fungus inducible PRP-1), EP 0 586 612 (nematode inducible), EP 0 712 273 (nematode inducible), WO 96/34949 (fungus inducible), PCT/EP96/02437 (nematode inducible) , EP 0 330 479 (stress inducible), US 5,510,474 (stress inducible), WO 96/12814 (cold inducible), EP 0 494 724 (tetracyclme inducible), EP 0 619 844 (ethylene inducible), EP 0 337 532 (salicylic acid inducible) , WO 95/24491 (thiamine inducible) and WO 92/19724 (light inducible) Other chemical inducible promoters are described m EP 0 674 608, EP 637 339, EP 455 667 and US ' *>

5 , 364 , 780 .

According to another embodiment of the invention, cells are transformed with constructs which inhibit the function of the endogenously expressed TPS or TPP. Inhibition of undesired endogenous enzyme activity is achieved m a number of ways, the choice of which is not critical to the invention. One method of inhibition of gene expression is achieved through the so-called 'antisense approach . Herein a DNA sequence is expressed which produces an RNA that is at least partially complementary to the RNA which encodes the enzymatic activity that is to be blocked. It is preferred to use homologous antisense genes as these are more efficient than heterologous genes An alternative method to block the synthesis of undesired enzymatic activities is the introduction mto the genome of the plant host of an additional copy of an endogenous gene present in the plant host It is often observed that such an additional copy of a gene silences the endogenous gene- this effect is referred to in the literature as the co-suppressive effect, or co-suppression. Details of the procedure of enhancing substrate availability are provided in the Examples of WO 95/01446, incorporated by reference herein. Host cells can be any cells in which the modification of hexokinase-signallmg can be achieved through alterations in the level of T-6-P. Thus, accordingly, all eukaryotic cells are subject to this invention. From an economic point of view the cells most suited for production of metabolic compounds are most suitable for the invention. These organisms are, amongst others, plants, animals, yeast, fungi.

However, also expression in specialized animal cells (like pancreatic beta-cells and fat cells) is envisaged

Preferred plant hosts among the Spermatophytae are the Angi ospermae , notably the Di cotyl edoneae, comprising inter al ia the Solanaceae as a representative family, and the Monocotyledoneae , comprising inter alia the Grammeae as a representative family Suitable host plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which contain a modified level of T-6-P, for instance by using recombinant DNA techniques to cause or enhance production of TPS or TPP in the desired plant or plant organ. Crops according to the invention include those which have flowers such as cauliflower { Brassi ca oleracea ) , artichoke ( Cynara scolymus ) , cut 2o flowers like carnation {Dianthus caryophylluε) , rose (Rosa spp) , Chrysanthemum, Petunia, Alstromeria, Gerbera, Gladiolus, lily (Lilium spp), hop (Humulus lupulus) , broccoli, potted plants like Rhododendron, Azalia, Dahlia, Begonia, Fuchsia, Geranium etc.; fruits such as apple {Malus, e.g. domesticus) , banana (Musa, e g. Acummata) , apricot { Prunus armeniaca) , olive {Oliva sativa) , pineapple {Ananas comosus) , coconut (Cocoε nucifera) , mango {Mangifera indica) , kiwi, avocado {Perεea americana) , berries (such as the currant, Ribeε, e.g rubrum) , cherries (such as the sweet cherry, Prunus, e g avium) , cucumber {Cucumiε, e.g. sativuε) , grape (Vitis, e g vinifera) , lemon (Citrus limon) , melon (Cucumis rnelo) , mustard {Smapiε alba and Braεεica mgra) , nuts (such as the walnut, Juglans, e.g. regia, peanut, Arachiε hypogeae) , orange {Citrus, e g. maxima), peach (Prunus, e g. perεica) , pear {Pyra, e g Communis) , pepper {Solanum, e g. capsicum) , plum {Prunus, e g domestica) , strawberry {Fragaπa, e g mo s chat a) , tomato (Lycopersicon, e.g eεculentum) , leaves, such as alfalfa {Medicago sativa), cabbages (such as Brasεica oleracea) , endive {Cichoreum, e.g endivia) , leek {Allium porrum) , lettuce {Lactuca sativa), spinach {Spmacia oleraceae) , tobacco {Nicotiana ta-bacum) , grasses like Festuca, Poa, rye-grass (such as Lolium perenne, Lolium multi florum and Arrenatherum εpp.), amenity grass, turf, seaweed, chicory {Cichoπum mtybus) , tea (Thea sinensiε) , celery, parsley {Petroεelmum crispum) , chevil and other herbs; roots, such as arrowroot {Maranta arundmacea) , beet (Beta vulgaπs) , carrot {Daucus carota), cassava {Mamhot esculenta) , ginseng {Panax ginseng) , turnip (Brasεica rapa) , radish (Raphanus sativus) , yam {Dioscorea eεculenta) , sweet potato (Ipomoea batatas), taro; seeds, such as beans (Phaεeoluε vulgaris) , pea (Piεum εativum) , soybean (Glycm max) , wheat (Triticum aestivum) , barley (Hordeum vulgare) , corn (Zea mays), rice {Oryza sativa) , bush beans and broad beans {Vicia faba) , cotton

{Gossypium εpp ), coffee (Coffea arabica and C. canephora) ; tubers, such as kohlrabi (Brassica oleraceae), potato (Solanum tujberosura) ; bulbous plants as onion (Allium cepa) , scallion, tulip (Tulipa spp ) , daffodil (Narcissus spp.), garlic (Allium sativum) ; stems such as cork-oak, sugarcane (Saccharum spp.), sisal (Sisal εpp.), flax (Lmum vulgare), jute; trees like rubber tree, oak (ρuercus spp.), beech (Betula εpp ), alder (Alnus spp.), ashtree (Acer spp.), elm ( Ulmus spp.), palms, ferns, ivies and the like 2. |

Transformation of yeast and fungal or animal cells can be done through normal state-of-the art transformation techniques through commonly known vector systems like pBluescript, pUC and viral vector systems like RSV and SV40 The method of introducing the expressible trehalose-phosphate synthase gene, the expressible trehalose-phosphate-phosphatase gene, or any other sense or antisense gene mto a recipient plant cell is not crucial, as long as the gene is expressed in said plant cell

Although some of the embodiments of the invention may not be practicable at preεent, e g becauεe some plant species are as yet recalcitrant to genetic transformation, the practicing of the invention m such plant species is merely a matter of time and not a matter of principle, because the amenability to genetic transformation as such is of no relevance to the underlying embodiment of the invention

Transformation of plant species is now routine for an impressive number of plant species, including both the Dicotyl edoneae as well as the Monocotyl edoneae In principle any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens et al (1982), Nature 296, 72, Negrutiu et al (1987), Plant Mol Biol 8, 363, electroporation of protoplasts (Shillito et al (1985) Bio/Technol 3_, 1099), microinjection into plant material (Crossway et al (1986) Mol Gen Genet 202), (DNA or RNA-coated) particle bombardment of various plant material (Klein et al (1987) , Nature 327, 70), infection with (non-integrative) viruses, in planta Agrobacteri um tumefaciens mediated gene transfer by infiltration of adult plants or transformation of mature pollen or microspores (EP 0 301 316) and the like A preferred method according to the invention comprises Agrobacterium-mediated DNA transfer Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S Patent 4,940,838).

Although considered somewhat more recalcitrant towards genetic transformation, monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material Presently, preferred methods for transformation of monocots are microprojectile bombardment of embryos, explants or suspension cells, and direct DNA uptake or (tissue) electroporation (Shimamoto et al . (1989), Nature 338, 274-276) Transgenic maize plants have been obtained by introducing the Streptomyces hygroεcopi cuε bar-gene, which encodes phosphinothπcm acetyltransferase (an enzyme which inactivates the herbicide phosphinothπcm) , mto embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon-Kamm (1990) , Plant Cell, 2, 603) The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee (1989), Plant Mol Biol. 13, 21) . Wheat plants have been regenerated from embryogenic suspension culture by selecting embryogenic callus for the establishment of the embryogenic suspension cultures (Vasil (1990) Bic/Technol. 8, 429) . The combination with transformation systems for these crops enables the application of the present invention to monocots.

Monocotyledonous plants, including commercially important crops such as rice and corn are also amenable to DNA transfer by Agrobacterium strains ( vide WO 94/00977; EP 0 159 418 Bl ; Gould et al (1991) Plant Physiol. 95, 426-434) . To obtain transgenic plants capable of constitutively expressing more than one chimeric gene, a number of alternatives are available including the following:

A. The use of DNA, e g a T-DNA on a binary plasmid, «;ιth a number of modified genes physically coupled to a second selectable marker gene The advantage of this method is that the chimeric genes are physically coupled and therefore migrate as a single Mendelian locus.

B. Cross-pollmation of transgenic plants each already capable of expressing one or more chimeric genes, preferably coupled to a selectable marker gene, with pollen from a transgenic plant which contains one or more chimeric genes coupled to another selectable marker. Afterwards the seed, which is obtained by this crossing, maybe selected on the basis of the presence of the two selectable markers, or on the basis of the presence of the chimeric genes themselves. The plants obtained from the selected seeds can afterwards be used for further crossing. In principle the chimeric genes are not on a single locus and the genes may therefore segregate as independent loci.

C. The use of a number of a plurality chimeric DNA molecules, e . g . plasmids, each havmg one or more chimeric genes and a selectable marker If the frequency of co-transformation is high, then selection on the basis of only one marker is sufficient In other cases, the selection on the basis of more than one marker is preferred D Consecutive transformation of transgenic plants already containing a first, second, (etc), chimeric gene with new chimeric DNA, optionally comprising a selectable marker gene. As in method B,the chimeric genes are in principle not on a single locus and the chimeric genes may therefore segregate as independent loci E Combinations of the above mentioned strategies The actual strategy may depend on several considerations as maybe easily determined such as the purpose of the parental lines (direct growing, use in a breeding programme, use to produce hybrids) but is not critical with respect to the described invention

It is known that practically all plants can be regenerated from cultured cells or tissues The means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided Shoots may be induced directly, or indirectly from callus via organogenesis or embryogenesis and subsequently rooted Next to the selectable marker, the culture media will generally contain various ammo acids and hormones, such as auxin and cytokmins It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa Efficient regeneration will depend on the medium, on the genotype and on the history of the culture If these three variables are controlled regeneration is usually reproducible and repeatable After stable incorporation of the transformed gene sequences mto the transgenic plants, the traits conferred by them can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed

Suitable DNA sequences for control of expression of the plant expressible genes (including marker genes), such as transcriptional initiation regions, enhancers, non-transcribed leaders and the like, may be derived from any gene that is expressed m a plant cell. Also intended are hybrid promoters combining functional portions of various promoters, or synthetic equivalents thereof Apart from constitutive promoters, inducible promoters, or promoters otherwise regulated m their expression pattern, e g developmentally or cell-type specific may be used to control expression of the expressible genes according to the invention.

To select or screen for transformed cells, it is preferred to include a marker gene linked to the plant expressible gene according to the invention to be transferred to a plant cell. The choice of a suitable marker gene in plant transformation is well within the scope of the average skilled worker; some examples of routinely used marker genes are the neomycin phosphotransferase genes conferring resistance to kanamycin (EP-B 131 623), the glutathion-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides

(EP-A 256 223), glutamine synthetase conferring upon overexpression resistance to glutamine synthetase inhibitors such aε phosphmothricm (WO 87/05327), the acetyl transferase gene from Streptomyceε viridochromogeneε conferring resistance to the selective agent phosphmothricm (EP-A 275 957), the gene encoding a

5-enolshιkιmate-3- phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycme, the bar gene conferring resistance agamst Bialaphos ( e . g . WO 91/02071) and the like The actual choice of the marker is not crucial as long as it is functional ( i . e . selective) in combination with the plant cells of choice.

The marker gene and the gene of interest do not have to be linked, since co-tranεformation of unlinked genes (U S. Patent 4,399,216) is also an efficient process m plant transformation. Preferred plant material for transformation, especially for dicotyledonous crops are leaf-discs which can be readily transformed and have good regenerative capability (Horsch et al (1985), Science 227, 1229) .

Specific use of the invention is envisaged in the following ways: as can be seen from the Examples the effects of the expression of TPP (which causes a decrease in the intracellular T-6-P concentration) are an increased leaf size, increased branching leading to an increase in the number of leaveε, increase in total leaf biomass, bleaching of mature leaves, formation of more small flowers and sterility. These effects are specifically useful m the following cases: increased leaf size (and increase in the number of leaves) is economically important for leafy vegetables such as spinach, lettuce, leek, alfalfa, silage maize; for ground coverage and weed control by grasses and garden plants; for crops in which the leaves are used as product, such as tobacco, tea, hemp and roses (perfumes') for the matting up of cabbage-like crops such as cauliflower

An additional advantage of the fact that these leaveε are stimulated in their metabolic activity is that they tend to burn all their intracellular resources, which means that they are low in starch-content For plants meant for consumption a reduction in starch content is advantageous in the light of the present tendency for low- calorie foodstuffs. Such a reduction in starch content also has effects on taste and texture of the leaves. An increase in the protein/carbohydrate balance as can be produced by the expression of TPP is especially important for leafy crops as silage maize

Increased branching, which is accompanied by a tendency to have stems with a larger diameter, can be advantageous in crops in which the stem is responsible for the generation of an economically attractive product Examples in this category are all trees for the increased production of wood, which is also a starting material for paper production, crops like hemp, sisal, flax which are used for the production of rope and linen, crops like bamboo and sugarcane, rubber- tree, cork-oak, for the prevention of flattening in crops or crop parts, like grains, corn, legumes and strawberries

A third phenomenon is increased bleaching of the leaves (caused by a decrease of photosynthetic activity) . Less colourful leaves are preferred for crops such as chicory and asparagus Also for cut flowers bleaching in the petals can be desired, for instance in Alεtromeria

An overall effect is the increase in biomass resulting from an increase in metabolic activity This means that the biomass consists of metabolized compounds such as proteins and fats Accordingly, there is an increased protein/carbohydrate balance in mature leaves which is an advantage for crops like silage maize, and all fodder which can be ensilaged. A similar increased protein/carbohydrate balance can be established in fruits, tubers and other edible plant parts

Outside the plant kingdom an increased metabolism would be beneficial for protein production in microorganisms or eukaryotic cell cultures. Both production of endogenous but also of heterologous proteins will be enhanced which means that the production of heterologous proteins in cultures of yeast or other unicellular organisms can be enhanced in this way For yeast this would give a more efficient fermentation, which would result m an increased alcohol yield, which of course is favourable in brewery processes, alcohol production and the like.

In animals or human beings it is envisaged that diseases caused by a defect in metabolism can be overcome by stable expression of TPP or TPS in the affected cells In human cells, the increased glucose consumption of many tumour cells depends to a large extent on the overexpression of hexokinase (Rempel et al (1996) FEBS Lett. 385, 233) . It is envisaged that the flux of glucose into the metabolism of cancer cells can be influenced by the expression of trehalose-6- phosphate synthesizing enzymes It has also been shown that the hexokinase activation is potentiated by the cAMP/PKA (protein kinase A pathway) Therefore, inactivation of this signal transduction pathway may affect glucose uptake and the proliferation of neoplasias Enzyme activities in mammalian cells able to synthesize trehalose-6-phosphate and trehalose and degrade trehalose have been shown m e g rabbit kidney cortex cells (Sacktor (1968) Proc Natl Acad.Sci USA 60, 1007) . Another example can be found in defects in insulin secretion in pancreatic beta-cells m which the production of glucose-6-phosphate catalyzed by hexokinase is the predominant reaction that couples rises m extracellular glucose levels to insulin secretion (Efrat et al (1994) , TIBS 19, 535) . An increase m hexokinase activity caused by a decrease of intracellular T-6-P then will stimulate insulin production m cells which are deficient in insulin secretion

Also in transgenic animals an increased protein/carbohydrate balance can be advantageous. Both the properties of on increased metabolism and an enhanced production of proteins are of large importance in farming in which animals should gam in flesh as soon as possible. Transformation of the enzyme TPP mto meat-producing animals like chickens, cattle, sheep, turkeys, goats, fish, lobster, crab, shrimps, snails etc, will yield animals that grow faster and have a more proteinaceous meat In the same way this increased metabolism means an increase m the burn rate of carbohydrates and it thus prevents obesity. More plant-specific effects from the decrease of intracellular T-6-P concentration are an increase in the number of flowers (although they do not seem to lead to the formation of seed) . However, an increase in the number of flowers is advantageous for cutflower plants and pot flower plants and also for all plants suitable for horticulture

A further effect of this flowering phenomenon is sterility, because the plants do not produce seed Sterile plants are advantageous m hybrid breeding. Another economically important aspect is the prohibiting of bolting of culture crops such as lettuce, endive and both recreational and fodder grasses This is a beneficial property because it enables the crop to grow without having to spend metabolic efforts to flowering and seed production Moreover, in crops like lettuce, endive and grasses the commercial product/application is non-bolted

Specific expression of TPP in certain parts (sinkε) of the plant can give additional beneficial effects It is envisaged that expression of TPP by a promoter which is active early m e g seed forming enables an increased growth of the developing seed A similar effect would be obtained by expressing TPP by a flower-specific promoter To put it shortly excessive growth of a certain plant part is possible if TPP is expressed by a suitable specific promoter. In fruits specific expression can lead to an increased growth of the skin in relation to the flesh This enables improvement of the peeling of the fruit, which can be advantageous for automatic peeling industries

Expression of TPP during the process of germination of oil- stormg seeds prevents oil-degradations. In the process of germination, the glyoxylate cycle is very active This metabolic pathway converts acetyl-CoA via malate into sucrose which can be transported and used as energy source during growth of the seedling Key-enzymes in this process are malate synthase and isocitrate lyase Expression of both enzymes is supposed to be regulated by hexokinase signalling. One of the indications for this regulation is that both 2- deoxyglucose and mannose are phosphorylated by hexokinase and able to transduce their signal, being reduction of malate synthase and isocitrate lyase expression, without being further metabolised Expression of TPP in the seed, thereby decreasing the inhibition of hexokinase, thereby inhibiting malate synthase and isocitrate lyase maintains the storage of oil mto the seeds and prevents germination.

In contrast to the effects of TPP the increase in T-6-P caused by the expression of TPS causes other effects as is illustrated in the Examples. From these it can be learnt that an increase in the amount of T-6-P causes dwarfing or stunted growth (especially at high expression of TPS), formation of more lancet-shaped leaves, darker colour due to an increase in chlorophyll and an increase m starch content. As is already acknowledged above, the introduction of an anti-sense trehalase construct will also stimulate similar effects as the introduction of TPS Therefore, the applications which are shown or indicated for TPS will equally be established by using as- trehalaεe Moreover, the use of double-constructs of TPS and as- trehalase enhances the effects of a single construct.

Dwarfing is a phenomenon that is desired m horticultural plants, of which the Japanese bonsai trees are a proverbial example However, also creation of mini-flowers in plants like allseed, roses, Amaryllis, Hortensia, birch and palm will have economic opportunities. Next to the plant kingdom dwarfing is also desired m animals. It is also possible to induce bolting m culture crops such as lettuce. This is beneficial because it enables a rapid production of seed. Ideally the expression of TPS for this effect should be under control of an inducible promoter.

Losε of apical dominance alεo causes formation of multiple shoots which is of economic importance for instance m alfalfa.

A reduction in growth is furthermore desired for the industry of "veggie snacks", m which vegetables are considered to be consumed in the form of snacks. Cherry-tomatoes is an example of redi ^~ed size vegetables which are successful in the market. It can be envisaged that also other vegetables like cabbages, cauliflower, carrot, beet and sweet potato and fruits like apple, pear, peach, melon, and several tropical fruits like mango and banana would be marketable on miniature size.

Reduced growth is desired for all cells that are detrimental to an organism, such as cells of pathogens and cancerous cells. In this last respect a role can be seen in regulation of the growth by changing the level of T-6-P. An increase in the T-6-P level would reduce growth and metabolism of cancer tissue. One way to increase the intracellular level of T-6-P is to knock-out the TPP gene of such cells by introducing a specific recombination event which causes the introduction of a mutation in the endogenous TPP-genes. One way in which this could be done is the introduction of a DNA-sequence able of introducing a mutation in the endogenous gene via a cancer cell specific internalizing antibody Another way is targeted microparticle bombardment with said DNA. Thirdly a cancer cell specific viral vectors having said DNA can be used.

The phenomenon of a darker green colour seen with an increased concentration of T-6-P, is a property which is desirable for pot flower plants and, in general, for species m horticulture and for recreational grasses.

Increase in the level of T-6-P also causes an increase in the storage carbohydrates such as starch and sucrose. This then would mean that tissues m which carbohydrates are stored would be able to store more material This can be illustrated by the Examples where it is shown that in plants increased biomass of storage organs such as tubers and thickened roots as in beets (storage of sucrose) are formed.

Crops in which this would be very advantageous are potato, sugarbeet, carrot, chicory and sugarcane. An additional economically important effect in potatoes is that after transformation with DNA encoding for the TPS gene (generating an increase in T-6-P) it has been found that the amount of soluble sugars decreases, even after harvest and storage of the tubers under cold conditions (4°C) . Normally even colder storage would be necessary to prevent early sprouting, but this results in excessive sweetening of the potatoes. Reduction of the amount of reducing sugars is of major importance for the food industry since sweetened potato tuber material is not suitable for processing because a Maillard reaction will take place between the reducing sugars and the ammo-acids which results in browning.

In the same way also inhibition of activity of mvertase can be obtained by transforming sugarbeets with a polynucleotide encoding for the enzyme TPS. Inhibition of invertase activity m sugarbeets after harvest is economically very important.

Also in fruits and seeds, storage can be altered This does not only result in an increased storage capacity but m a change in the composition of the stored compounds Crops in which improvements in yield m seed are especially important are maize, rice, cereals, pea, oilseed rape, sunflower, soybean and legumes Furthermore, all fruitbearmg plants are important for the application of developing a change in the amount and composition of stored carbohydrates. Especially for fruit the composition of stored products gives changes in solidity and firmness, which is especially important in soft fruits like tomato, banana, strawberry, peach, berries and grapes

In contrast to the effects seen with the expression of TPP, the expression of TPS reduces the ratio of protein/carbohydrate in leaves This effect is of importance in leafy crops such as fodder grasses and alfalfa. Furthermore, the leaves have a reduced biomass which can be of importance in amenity grasses, but, more important, they have a relatively increased energy content This property is especially beneficial for crops as onion, leek and εilage maize

Furthermore, also the viability of the seeds can be influenced by the level of intracellularly available T-6-P

Combinations of expression of TPP in one part of a plant and TPS in an other part of the plant can synergize to increase the above- described effects It is also possible to express the genes sequential during development by using specific promoters Lastly, it is also possible to induce expression of either of the genes involved by placing the coding the sequence under control of an inducible promoter. It is envisaged that combinations of the methods of application as described will be apparent to the person skilled m the art The invention is further illustrated by the following examples It is stressed that the Examples show specific embodiments of the inventions, but that it will be clear that variations on these examples and use of other plants or expression syεtems are covered by the invention. •3")

EXPERIMENTAL

DNA manipulations

All DNA procedures (DNA isolation from E. coli , restriction, ligation, transformation, etc ) are performed according to standard protocols (Sambrook et al (1989) Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, CSH, New York) .

Strains In all examples E.coli K-12 strain DH5α is used for cloning. The Agrobacteri um tumefacienε strains used for plant transformation experiments are EHA 105 and MOG 101 (Hood et al . (1993) Trans. Research 2, 208) .

Construction of Agrobacterium strain MOG101

Construction of Agrobacterium strain MOG101 is described in WO 96/21030.

Cloning of the E. coli otsA gene and construction of pMOG799 In E coli trehalose phosphate synthase (TPS) is encoded by the otsA gene located m the operon otsBA. The cloning and sequence determination of the otsA gene is described in detail in Example I of WO95/01446, herein incorporated by reference. To effectuate its expression m plant cells, the.open reading frame has been linked to the transcriptional regulatory elements of the CaMV 35S RNA promoter, the translational enhancer of the ALMV leader, and the transcriptional terminator of the nos-gene, as described in greater detail in Example I of WO95/01446, resulting in pMOG799. A sample of an E. coli strain harbouring pMOG799 has been deposited under the Budapest Treaty at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, P 0. Box 273, 3740 AG Baarn, The Netherlands, on Monday 23 August, 1993- the Accession Number given by the International Depositary Institution is CBS 430.93 Isolation of a patatm promoter/construction of PMOG546 A patatm promoter fragment is isolated from chromosomal DNA of Solanum_ tuberosum cv Bintje using the polymerase chain reaction. A set of oligonucleotides, complementary to the sequence of the upstream region of the λpat21 patatm gene (Bevan et al . (1986) Nucl. Acids Res. 14, 5564), is synthesized consisting of the following sequences.

5' AAG CTT ATG TTG CCA TAT AGA GTA G 3' PatB33.2 (SEQIDN0:5) 5' GTA GTT GCC ATG GTG CAA ATG TTC 3' PatATG 2 (SEQIDN0:6)

These primers are used to PCR amplify a DNA fragment of 1123bp, using chromosomal DNA isolated from potato cv. Bmtje as a template. The amplified fragment shows a high degree of similarity to the λpat21 patatm sequence and is cloned using EcoRI linkers into a pUClδ vector resulting in plasmid pMOG546

Construction of PMOG845

Construction of pMOG845 is described in WO 96/21030.

Construction of PVDH318, plastocvanm-TPS

Plasmid pMOG798 (described in WO95/01446) is digested with Hindlll and ligated with the oligonucleotide duplex TCVll and TCV12 (see construction of pMOG845) . The resulting vector is digested with Pstl and Hmdlll followed by the insertion of the PotPiII terminator resulting m pTCV118. Plasmid pTCV118 is digested with Smal and

HindiII yielding a DNA fragment comprising the TPS coding region and the PotPiII terminator. Bglll linkers were added and the resulting fragment was inserted m the plant binary expression vector pVDH275 (Fig. 1) digested with BamHI, yielding pVDH318 pVDH275 is a derivative of pMOG23 (Sijmons et al . (1990), Bio/Technol 8 217) harbouring the NPTII selection marker under control of the 35S CaMV promoter and an expression cassette comprising the pea plastocyanm (PC) promoter and nos terminator sequences. The plastocyanm promoter present in pVDH275 has been described by Pwee & Gray (1993) Plant J. 3, 437. This promoter has been transferred to the binary vector using PCR amplification and primers which contain εuitable cloning sites. Cloning of the E. coli otsB aene and construction of pMOGlOlO (35S CaMV TPP)

A set of oligonucleotides, TPP I (5' CTCAGATCTGGCCACAAA 3') (SEQ ID NO: 56) and TPP II (5' GTGCTCGTCTGCAGGTGC 3') (SEQ ID NO: 57), was synthesized complementary to the sequence of the E. coli TPP gene (SEQ ID NO: 3). These primers were used to PCR amplify a DNA fragment of 375bp harbouring the 3' part of the coding region of the E. coli TPP gene, introducing a Pstl site lObp down-stream of the stop codon, using pMOG748 (WO 95/01446) as a template. This PCR fragment was digested with Bglll and Pstl and cloned mto pMOG445 (EP 0 449 376 A2 example 7a) and linearized with Bglll and Pstl. The resulting vector was digested with Pstl and Hindlll and a PotPiII terminator was inserted (see construction pMOG845) The previous described vector was digested with Bglll and Hmdlll, the resulting 1325 bp fragment was isolated and cloned together with the 5'TPP PCRed fragment digested with Smal and Bglll mto pUClδ linearized with Smal and Hindlll. The resulting vector was called pTCV124. This vector was linearized with EcoRI and Smal and used to insert the 35S CaMV promoter (a 850bp EcoRI- 'Ncol' (the Ncol site was made blunt by treatment with mungbean nuclease) fragment isolated from pMOG18 containing the 35S CaMV double enhancer promoter) This vector was called ρTCV127. From this vector a 2.8kb EcoRI-Hindlll fragment was isolated containing the complete 35S TPP expression cassette and cloned in binary vector pMOG800 resulting in vector pMOGlOlO.

Construction of PVDH321, plastocyanm (PC) TPP

The BamHI site of plasmid pTCV124 was removed by BamHI digestion, filling-in and subsequent religation. Subsequent digestion with Hindlll and EcoRI yields a DNA fragment comprising the TPP coding region and the PotPiII terminator. BamHI linkers were added and the resulting fragment was inserted in the plant binary expression vector pVDH275 (digested with BamHI) yielding pVDH321. Construction of a patatm TPP expression vector

Similar to the construction of the patatm TPS expression vector (see construction of pMOG845), a patatm TPP expression vector was constructed yielding a binary vector (pMOG1128) which, after transformation, can effectuate expression of TPP in a tuber-specific manner.

Construction of other expression vectors

Similar to the construction of the above mentioned vectors, gene constructs can be made where different promoters are used, in combination with TPS, TPP or trehalase using binary vectors with the NPTII gene or the Hygromycm-resistance gene as selectable marker gene A description of binary vector pMOG22 harbouring a HPT selection marker is given in Goddijn et al . (1993) Plant J 4, 863

Triparental matmαs

The binary vectors are mobilized m triparental matmgs with the E. coli strain HB101 containing plasmid pRK2013 (Ditta et al (1980) Proc. Natl. Acad. Sci USA 77, 7347) into Agrobacterium t umefaciens strain MOG101 or EHA105 and used for transformation.

Transformation of tobacco ( Ni cotiana tabacum cv. SRI or cv Samsun NN) Tobacco was transformed by cocultivation of plant tissue with Agrobacteri um tumefaciens strain MOG101 containing the binary vector of interest as described Transformation was carried out using cocultivation of tobacco leaf disks as described by Horsch et al (1985) Science 227, 1229. Transgenic plants are regenerated from shoots that grow on selection medium containing kanamycin, rooted and transferred to soil.

Transformation of potato

Potato ( Solanum tuberosum cv Kardal) was transformed with the Agrobacterium strain EHA 105 containing the binary vector of interest. The basic culture medium was MS30R3 medium consisting of MS salts (Murashige and Skoog (1962) Physiol. Plant. 14, 473), R3 vitamins

(Ooms et al (1987) Theor. Appl. Genet. 73, 744), 30 g/1 sucrose, 0.5 g/1 MES with final pH 5.8 (adjusted with KOH) solidified when necessary with 8 g/1 Daichm agar. Tubers of Solanum tuberoεum cv Kardal were peeled and surface sterilized by burning them in 96% ethanol for 5 seconds. The flames were extinguished in sterile water and cut slices of approximately 2 mm thickness. Disks were cut with a bore from the vascular tissue and incubated for 20 minutes in MS30R3 medium containing 1-5 xlO⁸ bacteria/ml of Agrobacterium EHA 105 containing the binary vector. The tuber discs were washed with MS30R3 medium and transferred to solidified postculture medium (PM) . PM consisted of M30R3 medium supplemented with 3 5 mg/l zeatm riboside and 0.03 mg/l indole acetic acid (IAA) After two days, discs were transferred to fresh PM medium with 200 mg/l cefotaxim and 100 mg/l vancomycin. Three days later, the tuber discs were transferred to shoot induction medium (SIM) which consisted of PM medium with 250 mg/l carbenicillin and 100 mg/l kanamycin. After 4-8 weeks, shoots emerging from the discs were excised and placed on rooting medium (MS30R3-medium with 100 mg/l cefotaxim, 50 mg/l vancomycin and 50 mg/l kanamycin) . The shoots were propagated axenically by meristem cuttings.

Transformation of lettuce Transformation of lettuce, Lattuca sativa cv. Evola was performed according to Curtis et al. (1994) J. Exp. Bot. 45, 1441.

Transformation of sugarbeet

Transformation of sugarbeet, Beta vulgaπs (mamtamer population) was performed according to Fry et al. (1991) Third International Congress of ISPMB, Tucson USA Abstract No. 384, or according to Krens et al. (1996), Plant Sci. 116, 97.

Transformation of Lvcopersicon esculentum Tomato transformation was performed according to Van Roekel et al . (1993) Plant Cell Rep. 12, 644.

Transformation of Arabidopsis

Transformation of Arabidopsis thaliana was carried out either by the method described by Clarke et al. (1992) Plant. Mol. Biol. Rep. 10, 178 or by the method described by Valvekens et al . (1988) Proc. Natl. Acad. Sci. USA, 85, 5536. Induction of micro-tubers

Stem segments of m vi tro potato plants harbouring an auxiliary meristem were transferred to micro-tuber inducing medium Micro-tuber inducing medium contains 1 X MS-salts supplemented with R3 vitamins, 0.5 g/1 MES (final pH= 5.8, adjusted with KOH) and solidified with 8 g/1 Daichm agar, 60 g/1 sucrose and 2.5 mg/l kinetin. After 3 to 5 weeks of growth in the dark at 24°C, micro-tubers were formed.

Isolation of Validamvcm A Validamycin A has been found to be a highly specific inhibitor of trehalases from various sources ranging from (IC₅₀) 10-⁶M to 10-¹⁰M (Asano et al . (1987) J. Antibiot. 40, 526; Kameda et al (1987) J Antιbιot.40, 563) Except for trehalase, it does not significantly inhibit any α- or β-glycohydrolase activity. Validamycin A was isolated from Solacol, a commercial agricultural formulation (Takeda Chem. Induεt., Tokyo) as described by Kendall et al . (1990) Phytochemistry 29, 2525. The procedure involves ion-exchange chromatography (QAE-Sephadex A-25 (Pharmacia), bed vol 10 ml, equilibration buffer 0.2 mM Na-Pi pH 7) from a 3% agricultural formulation of Solacol. Loading 1 ml of Solacol on the column and eluting with water in 7 fractions, practically all Validamycin was recovered in fraction 4. Based on a 100% recovery, using this procedure, the concentration of Validamycin A was adjusted to 1.10^"3 M in MS-medium, for use in trehalose accumulation tests Alternatively, Validamycin A and B may be purified directly from Streptomyces hygroscopi cus var. limoneus , as deεcribed by Iwasa et al. (1971) J. Antibiot. 24, 119, the content of which is incorporated herein by reference.

Carbohydrate analysis

Carbohydrates were determined quantitatively by anion exchange chromatography with pulsed electrochemical detection. Extracts were prepared by extracting homogenized frozen material with 80% EtOH After extraction for 15 minutes at room temperature, the soluble fraction is evaporated and dissolved in distilled water. Samples ⁽25 μl) were analyzed on a Dionex DX-300 liquid chromatograph equipped with a 4 x 250 mm Dionex 35391 carbopac PA-1 column and a 4 x 50 mm Dionex 43096 carbopac PA-1 precolumn. Elution was with 100 mM NaOH at 1 ml/mm followed by a NaAc gradient. Sugars were detected with a pulsed electrochemical detector (Dionex, PED) . Commercially available carbohydrates (Sigma) were used as a standard.

Starch analysis

Starch analysis was performed as described in: Aman et al . (1994) Methods m Carbohydrate Chemistry, Volume X (eds. BeMiller et al . ) , pp 111-115.

Expression analysis

The expression of genes introduced in various plant species was monitored using Northern blot analysis.

Trehalose-6-phosphate phosphatase assay TPP was assayed at 37°C by measuring the production of [¹⁴C]trehalose from [¹⁴C] trehalose-6-phosphate (Londesborough and Vuorio (1991) J. of Gen. Microbiol. 137, 323) . Crude extracts were prepared in 25 mM Tris, HCI pH 7.4, containing 5.5 mM MgCl₂• Samples were diluted to a protein concentration of 1 mg/ml in extraction buffer containing 1 mg/ml BSA. Standard assay mixtures (50 μl final volume) contained 27.5 mM Tris, HCI pH 7.4, 5.5 mM MgCl₂, 1 mg/ml BSA and 0.55 mM T-6-P (specific activity 854 cpm/nmol). Reactions were initiated by the addition of 5μl enzyme and terminated after 1 hour by heating for 5 minutes in boiling water AG1-X8 (formate) anion-exchange resin (BioRad) was added and the reaction mixtures were centrifuged after 20 minutes of equilibration at room temperature. The radioactivity in the supernatant of the samples (400 μl) was measured by liquid scintillation counting.

Preparation of plant extracts for hexokinase assays

Frozen plant material was grinded in liquid nitrogen and homogenized for 30 seconds with extraction buffer (EB lOOmM HEPES pH7.0 (KOH), 1% (w/v) PVP, 5mM MgCl₂, 1.5 mM EDTA, 0.1 %v/v β-MeOH) including Proteinase Inhibitors Complete (Boehringer Mannheim) . After centrifugation, proteins in the supernatant were precipitated using 80% ammoniumsulphate and dissolved in Tris-HCl pH 7.4 and the extract was dialyzed overnight against lOOmM Tris-HCl pH 7.4. Part of the sample was used in the hexokinase assay. 3fS>

Hexokinase assay

Hexokinase activity was measured in an assay containing 0.1 M Hepes- KOH pH 7.0, 4 mM MgCl_2, 5 mM ATP, 0.2 mM NADP⁺, 10 U/ml Creatme Phosphate Kmase (diεεolved in 50% glycerol, 0.1% BSA, 50 mM Hepeε pH 7.0) , 3.5 mM Creatme Phosphate, 7 U/ml Glucose-6-Phosphate

Dehydrogenase and 2 mM Glucose by measuring the increase in OD at 340 nm at 25 °C.

When 2 mM Fructose was used instead of glucose as substrate for the hexokinase reaction, 3 8 U/ml Phosphoglucose Isomerase was mcluded. Alternatively, a hexokinase assay as described by Gancedo et al . (1977) J. Biol. Chem. 252, 4443 was used.

EXAMPLE 1 Expression of the E. coli otsA gene (TPS) in tobacco and potato

Transgenic tobacco plants were generated harbouring the otsA gene driven by the de35SCaMV promoter (pMOG799) or the plastocyanm promoter (pVDH318).

Transgenic potato plants were generated harbouring the otsA gene driven by the potato tuber-specific patatm promoter (pMOG845) .

Tobacco leaf discs were transformed with the binary vector pMOG799 using Agrobacterium tumefaci ens . Transgenic shoots were selected on kanamycin. Leaves of some soil-grown plantε did not fully expand in lateral direction, leading to a lancet-shaped morphology (Fig. 31) . Furthermore, apical dominance was reduced resulting in stunted growth and formation of several axillary shoots. Seven out of thirty-two plants showed severe growth reduction, reaching plant heights of 4-30 cm at the time of flowering (Table 1) ^

Table 1 . Trehalose accumulation in leaf samples of otsA transgenic tobacco plants and their plant length at the time of flowering.

ND: not determined

Control plants reached lengths of 60-70 cm at the time of flowering. Less seed was produced by transgenic lines with the stunted growth phenotype. Northern blot analysis confirmed that plants having the stunted growth phenotype expressed the otsA gene from E . coli (Fig. 2) . In control plants no transcript could be detected. The functionality of the introduced gene was proven by carbohydrate analyses of leaf material from 32 transgenic greenhouse-grown tobacco plants, revealing the presence of 0.02 to 0.12 mg.g^-1 fresh weight trehalose in plants reduced in length (table 1) indicating that the product of the TPS- catalyzed reaction is dephosphorylated by plant phosphatases. Further proof for the accumulation of trehalose in tobacco was obtained by treating crude extracts with porcine trehalase. Prolonged incubation of a tobacco leaf extract with trehalase resulted in complete degradation of trehalose (data not shown) . Trehalose was not detected in control plants or transgenic tobacco plants without an aberrant phenotype. 4θ

Table la. Primary PC-TPS tobacco transformants

Transgenic pVDH318 transgenic tobacco plants developed stunted growth and development of small leaves which were darker green and slightly thicker than control leaves, a phenotype similar to the pMOG799 transgenic plants (table la) . Further analysis of these leaves showed an increased fresh and dry weight per leaf-area compared to the controls (table la and 2). The dark green leaves indicate the presence of more chlorophyll in the transgenic leaves (table lb) . Plants transgenic for pMOG799 (35STPS) and pMOG1177 (PCTPS) were analyzed on soluble carbohydrates, chlorophyll, trehalose and starch (Fig. 32) . pMOG1177 is functionally identical to pVDH318.

Table lb. Chlorophyll content of N. tabacum leaves (T₀) transgenic for PC-TPS

Note: light conditions during growth will influence the determined levels of chlorophyll significantly. The calculated amounts of chlorophyll may thus only be compared between plants harvested and analyzed within one experiment! Table 2 . Fresh weight and dry weight data of leaf material transgenic for plastocyanιn-TPS_{E co}ι

N. tabacum cv. Samsun NN transgenic for PC-TPS

Calculation of the ratio between the length and width of the developing leaves clearly indicate that leaves of plants transgenic for PC-TPS are more lancet-shaped (table 3).

Potato Solanum tuberosum cv. Kardal tuber discs were transformed with Agrobacteri um tumefaci ens EHA105 harbouring the binary vector pMOG845 Transgemcs were obtained with transformation frequencies comparable to empty vector controls. All plants obtained were phenotypically indistinguishable from wild type plants indicating that use of a tissue specific promoter prevents the phenotypes observed in plants where a constitutive promoter drives the TPS gene. Micro-tubers were induced on stem segments of transgenic and wild-type plants cultured on microtuber-mducmg medium supplemented with IO"³ M Validamycin A As a control, microtubers were induced on medium without Validamycin A. Microtubers induced on medium with Validamycin A showed elevated levels of trehalose in comparison with microtubers grown on medium without Validamycin A (table 4) . The presence of small amounts of trehalose m wild-type plants indicates the presence of a functional trehalose biosynthetic pathway. +3

Table 3 Tobacco plants (cv. Samsun NN) transgenic for pVDH318

typical TPS phenotypes Ratio 1/w average of controls is 1.50 M"

Table 4 . Trehalose (% fresh weight)

EXAMPLE 2

Expression of the E. coli otsB gene (TPP) in tobacco

Transgenic tobacco plants were generated harbouring the otsB gene driven by the double enhanced 35SCaMV promoter (pMOGlOlO) and the plastocyanm promoter (pVDH321). Tobacco plants (cv. Samsun NN) transformed with pMOGlOlO revealed in the greenhouse the development of very large leaves (leaf area increased on average up to approximately 140%) which started to develop chlorosis when fully developed (Fig. 31) . Additionally, thicker stems were formed as compared to the controls, m some instances leading to bursting of the stems. In some cases, multiple stems were formed (branching) from the base of the plant (table 5) Leaf samples of plants developing large leaves revealed 5-10 times enhanced trehalose-6-phosphate phosphatase activities compared to control plants proving functionality of the gene introduced. The dry and fresh weight/cm² of the abnormal large leaves was comparable to control leaves, indicating that the increase in size is due to an increase in dry matter and not to an increased water content. The inflorescence was also affected by the expression of TPP. Plants which had a stunted phenotype, probably caused by the constitutive expression of the TPP gene in all plant parts, developed many small flowers which did not fully mature and fell off or necrotized. The development of flowers and seed setting seems to be less affected m plants which were less stunted. -f5

wt = wild- type V

Tobacco plants (cv. Samsun NN) transformed with pVDH321 revealed m the greenhouse a pattern of development comparable to pMOGlOlO transgenic plants (table 6) .

Plants transgenic for pMOGlOlO (35S-TPP) and pMOGH24 (PC-TPP) were analyzed on carbohydrates, chlorophyll, trehalose and starch (Fig. 32) . For chlorophyll data see also Table 6a.

Table 6a. Chlorophyll content of N tabacum leaves (T₀) transgenic for PC-TPP

Note: light conditions during growth will influence the determined levels of chlorophyll significantly. The calculated amounts of chlorophyll may thus only be compared between plants harvested and analyzed withm one experiment¹ EXAMPLE 3

Isolation of gene fragments encoding trehalose-6-phosphate synthases from Selaginβlla lepidophylla and Hβl i anthus annuus Comparison of the TPS protein sequences from E. col i and S. cerevisiae revealed the presence of several conserved regions. These regions were used to design degenerated primers which were tested in PCR amplification reactions using genomic DNA of E. coli and yeast as a template A PCR program was used with a temperature ramp between the annealing and elongation step to facilitate annealing of the degenerate primers

PCR amplification was performed using primer sets TPSdeg 1/5 and TPSdeg 2/5 using cDNA of Selagmel la l epidophyl la as a template.

Degenerated primers used (IUB code)

TPSdegl GAY ITI ATI TGG RTI CAY GAY TAY CA (SEQ ID NO:7)

TPSdeg2 : TIG GIT KIT TYY TIC AYA YIC CIT TYC C (SEQ ID NO: 8) TPSdeg5- GYI ACI ARR TTC ATI CCR TCI C (SEQ ID NO:9)

PCR fragments of the expected size were cloned and sequenced Since a large number of homologous sequences were isolated, Southern blot analysis was used to determine which clones hybridized with Selagmel l a genomic DNA. Two clones were isolated, clone 8 of which the sequence is given in SEQ ID NO 42 (PCR primer combination 1/5) and clone 43 of which the sequence is given in SEQ ID NO 44 (PCR primer combination 2/5) which on the level of amino acids revealed regions with a high percentage of identity to the TPS genes from E. col i and yeast. One TPS gene fragment was isolated from Helianthus annuus (sunflower) using primer combination TPSdeg 2/5 in a PCR amplification with genomic DNA of H. annuuε as a template. Sequence and Southern blot analysis confirmed the homology with the TPS genes from E. coli , yeast and Selagmella . Comparison of these sequenceε with EST sequences (expressed sequence tags) from various organisms, see Table 6b and SEQ ID NOS 45-53 and 41, indicated the presence of highly homologous genes in rice and Arabidopsis , which supports our mvention that most plants contain TPS homologous genes (Fig. 3) . ^,Λ>

Table 6b.

5o

EXAMPLE 4 Isolation of plant TPS and TPP genes from Ni cotiana tabacum

Fragments of plant TPS- and TPP-encodmg cDNA were isolated usmg PCR on cDNA derived from tobacco leaf total RNA preparations The column "nested" in table 7 indicates if a second round of PCR amplification was necessary with primer set 3 and 4 to obtain the corresponding DNA fragment. Primers have been included in the sequence listing (table 7) . Subclonmg and subsequent sequence analysis of the DNA fragments obtained with the primer sets mentioned revealed subεtantial homology to known TPS geneε (Fig 4 & 5) .

Table 7. Amplification of plant derived TPS and TPP cDNAs

S I

EXAMPLE 5 Isolation of a bipartite TPS/TPP gene from He l i an thus annuus and Nicotiana tabacum

Using the sequence information of the TPS gene fragment from sunflower ( Hel ianthus annuus) , a full length sunflower TPS clone was obtained using RACE-PCR technology.

Sequence analysis of this full length clone and alignment with TPS2 from yeast (Fig. 6) and TPS and TPP encoding sequences indicated the isolated clone encodes a TPS/TPP bipartite enzyme (SEQ ID NO 24, 26 and 28) . The bipartite clone isolated (pMOG1192) was deposited at the Central Bureau for Strain collections under the rules of the Budapeεt treaty with accession number CBS692.97 at April 21, 1997. Subsequently, we investigated if other plant species also contain TPS/TPP bipartite clones. A bipartite TPS/TPP cDNA was amplified from tobacco. A DNA product of the expected size ( i . e . 1.5 kb) was detected after PCR with primers TPS degl/TRE-TPP-16 and nested with TPS deg2/TRE-TPP-15 (SEQ ID NO: 33). An identical band appeared with PCR with TPS degl/TRE-TPP-6 (SEQ ID NO: 34) and neεted with TPS deg2/TRE- TPP-15 The latter fragment was shown to hybridize to the sunflower bipartite cDNA in a Southern blot experiment. Additionally, using computer database searches, an Arabidopεis bipartite clone was identified (SEQ ID NO: 39)

EXAMPLE 6 Expression of plant derived TPS genes in plants

Further proof for the function of the TPS genes from sunflower and Sel agmel la lepidophylla was obtained by isolating their correspondmg full-length cDNA clones and subsequent expression of these clones in plants under control of the 35S CaMV promoter. Accumulation of trehalose by expression of the Seliganella enzyme has been reported by Zentella and Itumaga (1996) (Plant Physiol. Ill, Abstract 88)

EXAMPLE 7

Genes encoding TPS and TPP from monocot species A computer search in Genbank sequences revealed the presence of several rice EST-sequences homologous to TPSl and TPΞ2 from yeast (Fig. 7) which are included in the sequence listing (SEQ ID NO: 41,51,52 and 53) . EXAMPLE 8 Isolation human TPS gene

A TPS gene was isolated from human cDNA. A PCR reaction was performed on human cDNA using the degenerated TPS primers deg2 and deg5 This led to the expected TPS fragment of 0.6 kb Sequence analysis (SEQ ID NO.10) and comparison with the TPSyeast sequence indicated that isolated sequence encodes a homologous TPS protein (Fig. 8)

EXAMPLE 9 Inhibition of endogenous TPS expression by anti-sense inhibition

The expression of endogenous TPS genes can be inhibited by tne anti- sense expression of a homologous TPS gene under control of promoter sequences which drive the expression of such an anti-sense TPS gene in cells or tissue where the inhibition is desired For this approach, it is preferred to use a fully identical sequence to the TPS gene which has to be suppressed although it is not necesεary to express the entire coding region in an anti-sense expression vector. Fragments of such a coding region have also shown to be functional in the anti- sense inhibition of gene-expression. Alternatively, heterologous genes can be used for the anti-sense approach when these are sufficiently homologous to the endogenous gene

Binary vectors similar to pMOG845 and pMOGlOlO can be used ensuring that the coding regions of the introduced genes which are to be suppressed are introduced in the reverse orientation. All promoters which are suitable to drive expression of genes m target tissues are also suitable for the anti-sense expression of genes

EXAMPLE 1& Inhibition of endogenous TPP expression by anti-sense inhibition

Similar to the construction of vectors which can be used to drive anti-sense expression of tps m cells and tissues (Example 9), vectors can be constructed which drive the anti-sense expression of TPP genes EXAMPLE 11

Trehalose accumulation in wild-type tobacco and potato plants grown on Validamycin A

Evidence for the presence of a trehalose biosynthesis pathway in tobacco was obtained by culturing wild-type plants in the presence of 10^_3M of the trehalase inhibitor Validamycin A. The treated plants accumulated very small amounts of trehalose, up to 0.0021% (fw). Trehalose accumulation was never detected in any control plants cultured without inhibitor Similar data were obtained with wild-type microtubers cultured m the presence of Validamycin A Ten out of seventeen lines accumulated on average 0.001% trehalose (fw) (table 4) No trehalose was observed in microtubers which were induced on medium without Validamycin A.

EXAMPLE 12

Trehalose accumulation in potato plants transgenic for as- trehalase

Further proof for the presence of endogenous trehalose biosynthesis genes was obtained by transforming wild-type potato plants with a 35S CaMV anti-sense trehalase construct (SEQ ID NO: 54 and 55, pMOG1027; described in WO 96/21030) A potato shoot transgenic for pMOG1027 showed to accumulate trehalose up to 0.008% on a fresh weight basis The identity of the trehalose peak observed was confirmed by specificly breaking down the accumulated trehalose with the enzyme trehalase Tubers of some pMOG1027 transgenic lines showed to accumulate small amounts of trehalose (Fig. 9)

EXAMPLE 13 Inhibition of plant hexokinase activity by trehalose-6- phosphate

To demonstrate the regulatory effect of trehalose-6-phosphate on hexokinase activity, plant extracts were prepared and teεted for hexokinase activity m the absence and preεence of trehalose-6- phosphate. • Potato tuber extracts were assayed using fructose (Fig. 10, Fig. 11) and glucose (Fig. 11) as substrate. The potato tuber assay using 1 mM T-6-P and fructose as substrate was performed according to Gancedo et al . (1997) J. Biol. Chem. 252, 4443. The following assays on tobacco, rice and maize were performed according to the assay described in the 97/ 497

⁵+ section experimental.

• Tobacco leaf extracts were assayed using fructose (Fig. 12) and glucose (Fig. 12, Fig 13) as substrate.

^• Rice leaf extracts were assayed using fructose and glucose (Fig. 14) as substrate.

• Maize leaf extracts were assayed using fructose and glucose (Fig. 15) as substrate.

EXAMPLE 14 Inhibition of hexokinase activity in animal cell cultures by trehalose-6-phosphate

To demonstrate the regulation of hexokinase activity in animal cells, total cell extracts were prepared from mouse hybridoma cell cultures A hexokinase assay was performed using glucose or fructose as substrate under conditions as described by Gancedo et al . (see above) . Mouse hybridoma cells were subjected to osmotic shock by exposing a cell pellet to 20% sucrose, followed by distilled water This crude protein extract was used in the hexokinase assay (50 μl extract corresponding to ca.200 μg protein) .

Table 8 . Inhibition of animal hexokinase activity by T-6-P

The data obtained clearly showed that hexokinase activity m mouse cell extracts is inhibited by trehalose-6-phosphate The T-6-P concentration range in which this effect is noted is comparable to what has been observed m crude plant extracts No difference is noted in the efficiency of hexokinase inhibition by trehalose-6-phosphate using glucose or fructose as substrate for the enzyme

EXAMPLE 15 Photosynthesis and respiration of TPS and TPP expressing tobacco plants

Using tobacco plants transgenic for 35S-TPP (1010-5), PC-TPS (1318-10 and 1318-37) and wild-type Samsun NN plants, effects of expression of these genes on photosynthesis and respiration were determined in leaves

Measurements were performed m a gas exchange-experimental set-up Velocities of gas-exchange were calculated on the basis of differences in concentration between ingoing and outgoing air using infra-red gas- analytical equipment Photosynthesis and respiration were measured from identical leaves From each transgenic plant, the youngest, fully matured leaf was used (upper-leaf) and a leaf that was 3-4 leaf- "stores" lower (lower-leaf)

Photosynthesis was measured as a function of the photosynthetic active light intensity (PAR) from 0-975 μmol m 2 _s ι (200 Watt m 2), m four¬ fold at C0₂-concentrations of 350 vpm and 950 vpm

Respiration was measured using two different time-scales. Measurements performed during a short dark-period after the photosynthesis experiments are coded RD m table 9. These values reflect instantaneous activity since respiration varies substantially during the dark-period Therefor, the values for the entire night-period were also summed as shown in table 10 (only measured at 350 vpm C0₂)

Table 9. Rate of photosynthesis and respiration, STD is standard deviation

V

Table 10. Respiration during 12 hour dark period (mmol C0₂) STD is standard deviation

In contrast to the respiration in the upper-leaves, m lower leaves the respiration of TPS transgenic plants is significantly higher than for wild-type and TPP plants (table 10) indicating a higher metabolic activity. The decline in respiration during aging of the leaves is significantly less for TPS transgenic plants.

Also, the photosynthetic characteristics differed significantly between on the one hand TPS transgenic plants and on the other hand TPP transgenic and wild-type control plants. The AMAX values (maximum of photosynthesis at light saturation) , efficiency of photosynthesis (EFF) and the respiration velocity during a short dark-period after the photosynthetic measurements (RD) are shown in table 9. On average, the upper TPS leaves had a 35% higher AMAX value compared to the TPP and wild-type leaves. The lower leaves show even a higher increased rate of photosynthesis (88%).

To exclude that differences in light-absorption were causing the different photosynthetic rates, absorption values were measured with a SPAD-502 (Minolta) . No significant differences in absorption were measured (table 11) . 5<5

Table 11. Absorbtion values of transgenic lines

EXAMPLE 1£

Chlorophyll-fluorescence of TPS and TPP expressing tobacco plants

Using tobacco plants transgenic for 35S-TPP (1010-5), PC-TPS (1318-10 and 1318-37) and wild-type Samsun NN plants, effects of expressing these genes were determined on chlorophyll fluorescence of leaf material Two characteristics of fluorescence were measured:

1) ETE (electron transport efficiency), as a measure for the electron transport velocity and the generation of reducing power, and

2) Non-photochemical quenching, a measure for energy-dissipation caused by the accumulation of assimilates.

Plants were grown m a greenhouse with additional light of 100 μmol. m-2.5-¹ (04:00 - 20:00 hours) Day/night T=21-C/18°C; R.H. ± 75%.During a night-period preceding the measurements (duration 16 hours) , two plants of each genotype were transferred to the dark and two plants to the light (±430 μmol m-z.s"¹, 20°C, R.H. 70%) . The youngest fully matured leaf was measured. The photochemical efficiency of PSII (photosystem II) and the "non-photochemical quenching" parameters were determined as a function of increasing, light intensity. At each light intensity, a 300 sec. stabilisation time was taken. Measurements were performed at 5, 38, 236, 422 and 784,μmol m^.s"¹ PAR with a frequency of 3 light-flashes mm-i, 350 ppm C0₂ and 20% 0₂ Experiments were replicated using identical plants, reversing the pretreatment from dark to light and vice versa. The fluorescence characteristics are depicted in Fig. 16. ^>ϋ)

The decrease in electron-transport efficiency (ETE) was comparable between TPP and wild-type plants TPS plants clearly responded less to a increase of light intensity. This difference was most clear in the light pretreatment These observations are in agreement with the "non- photochemical " quenching data TPS plants clearly responded less to the additional supply of assimilates by light compared to TPP and wild-type plants. In the case of TPS plants, the negative regulation of accumulating assimilates on photosynthesis was significantly reduced

EXAMPLE 17

Export and allocation of assimilates in TPS and RPP expressing tobacco plants

Using tobacco plants transgenic for 35S-TPP (1010-5) and PC-TPS (1318- 37) ,

1) the export of carbon-assimilates from a fully grown leaf (indicating "relative source activity", Koch (1996) Annu Rev Plant Physiol Plant. Mol. Biol. 47, 509 and

2) the net accumulation of photo-assimilates m εmks ("relative sink activity") , during a light and a dark-period, were determined

Developmental stage of the plants flowerbuds just visible Labelling technique used: Steady-state high abundance 13C-labellmg of photosynthetic products (De Visser et al (1997) Plant Cell Environ 20, 37) . Of both genotypes, 8 plants, using a fully grown leaf, were labelled with 5.1 atom% ¹³Cθ2 during a light-period (10 hours) , when appropriate followed by a dark-period (14 hours) . After labelling, plants were split in: 1) shoot-tip, 2) young growing leaf, 3) young fully developed leaf (above the leaf being labelled) , 4) young stem (above the leaf being labelled), 5) labelled leaf, 6) petiole and base of labelled leaf, 7) old, senescing leaf, 8) other and oldest leaves lower than the labelled leaf, 9) stem lower than the labelled leaf, 10) root-tips Number, fresh and dry weight and ¹³C percentage (atom % ¹³C) of carbon were determined Next to general parameters as biomass, dry matter and number of leaves, calculated were 1) Export of C out of the labelled leaf; 2) the relative contribution of imported C in plant parts, 3) the absolute amount of imported C in plant parts, 4) the relative distribution of imported C during a light period and a complete light and dark-period. ^o

The biomass above soil of the TPP transgenics was 27% larger compared to the TPS transgenics (P<0.001); also the root-system of the TPP transgenics were better developed. The TPP plants revealed a significant altered dry matter distribution, +39% leaf and +10% stem biomass compared to TPS plants. TPS plants had a larger number of leaves, but a smaller leaf-area per leaf. Total leaf area per TPS plant was comparable with wild-type (0.4 m² plant-¹)

- Relative source activity of a fully developed leaf The net export rate of photosynthates out of the labelled leaf is determined by the relative decrease of the % "new C" during the night (for TPP 39% and for TPS 56%) and by the total fixated amount present m the plant using the amount of "new C" in the plant (without the labelled leaf) as a measure After a light period, TPP leaves exported 37% compared to 51% for TPS leaves (table 11) . In a following dark- period, this percentage increased to respectively 52% and 81%. Both methods support the conclusion that TPS transgenic plants have a significantly enhanced export rate of photosynthetic products compared to the TPP transgenic plants

- Absolute amount of "new C" in plant parts

Export by TPS transgenics was significantly higher compared to TPP transgenics Young growing TPS leaves import C stronger compared to young growing TPP leaves.

- Relative increase of "new C" m plant parts: smk-strenσth The relative contribution of "new C" to the concerning plant part is depicted in Fig. 17. This percentage is a measure for the sink- strength. A significant higher sink-strength was present in the TPS transgenics, especially in the shoot-top, the stem above and beneath the labelled leaf and the petiole of the labelled leaf. /,

Table 11. Source activity of a full grown labelled leaf: C accumulation and -export. Nett daily accumulation and export of C-assimilates in labelled leaf and the whole plant (above soil) after steady-state 13^c-labelling during a light period (day) . N=4: LSD values indicated the smallest significant differences for P<0.05

- Relative distribution, within the plant, of "new C" between the plant parts: relative sink strength

The distribution of fixed carbon between plant organs (Fig. 18) confirmed the above mentioned conclusions. TPS transgenic plants revealed a relative large export of assimilates to the shoot-top, the young growing leaf (day) and even the oldest leaf (without axillary meristems) , and to the young and old stem.

EXAMPLE 18; Lettuce Performance of lettuce plants transgenic for PC-TPS and PC- TPP

Constructs used in lettuce transformation experiments: PC-TPS and PC- TPP. PC-TPS transgenics were rescued during regeneration by culturing explants on 60 g/1 sucrose. The phenotypes of both TPS and TPP transgenic plants are clearly distinguishable from wild-type controls; TPS transgenic plants have thick, dark-green leaves and TPP transgenic plants have light-green leaves with a smoother leaf-edge when compared to wild-type plants. The morphology of the leaves, and most prominent the leaf-edges, was clearly affected by the expression of TPS and TPP. Leaves transgenic for PC-TPS were far more "notched" than the PC-TPP transgenic leaves that had a more smooth and round morphology (Fig. 19). Leaf extracts of transgenic lettuce lines were analyzed for sugars and starch (Fig. 20) .

EXAMPLE 19: Sugarbeet Performance of sugarbeet plants transgenic for PC-TPS and PC-TPP

Constructs used in sugarbeet transformation experiments: PC-TPS and PC-TPP. Transformation frequencies obtained with both the TPS and the TPP construct were comparable to controls. The phenotypes of both TPS and TPP transgenic plants were clearly distinguishable from wild-type controls; TPS transgenic plants had thick, dark-green leaves and TPP transgenic plants had light-green coloured leaves with slightly taller petioles when compared to wild-type plants (Fig. 21). Taproot diameter was determined for all plants after ca. 8 weeks of growth in the greenhouse Some PC-TPS transgenic lines having a leaf size similar to the control plants showed a significant larger diameter of the tap¬ root (Fig. 22) . PC-TPP transgenic lines formed a smaller taproot compared to the non-transgenic controls. Leaf extracts of transgenic sugarbeet lines were analyzed for sugars and starch (Fig. 20) .

EXAMPLE 20: Arabidopsis

Performance of Arabi dopsi s plants transgenic for PC-TPS and

PC-TPP

Constructs used in Arabidopsiε transformation experiments: PC-TPS and PC-TPP. The phenotypes of both TPS and TPP transgenic plants were clearly distinguishable from wild-type controls; TPS transgenic plants had thick, dark-green leaves and TPP transgenic plants had larger, bleaching leaves when compared to wild-type plants Plants with high levels of TPP expression did not set seed. EXAMPLE 21: Potato Performance of Solanum tuberosum plants transgenic for TPS and TPP constructs

Construct. 35S-TPS pMOG799

Plants transgenic for pMOG799 were grown m the greenhouse and tuber- yield was determined (Fig. 23) . The majority of the transgenic plants showed smaller leaf sizes when compared to wild-type controls. Plants with smaller leaf-sizes yielded less tuber-mass compared to control lines (Fig. 25) .

Construct: 35S-TPP pMOGlOlO and PC-TPP pMOG1124 Plants transgenic for pMOG 1010 and pMOG1124 were grown m the greenhouse and tuber-yield was determined Tuber-yield (Fig. 24) was comparable or less than the wild-type control lines (Fig. 25) .

Construct: PC-TPS pMOG1093

Plants transgenic for pMOG1093 were grown in the greenhouse and tuber- yield was determined. A number of transgenic lines having leaves with a size comparable to wild-type (B-C) and that were slightly darker green in colour yielded more tuber-mass compared to control plants (Fig. 26) Plants with leaf sizes smaller (D-G) than control plants yielded less tuber-mass.

Construct: Pat-TPP pMOG1128

Microtubers were induced m vi tro on explants of pat-TPP transgenic plants. The average fresh weight biomass of the microtubers formed was substantially lower compared to the control lines

Construct- Pat-TPS pMOG845

Plants transgenic for pMOG 845 were grown m the greenhouse and tuber- yield was determined. Three Pat-TPS lines produced more tuber-mass compared to control lines (Fig. 27)

Construct: PC TPS Pat TPS; pMOG1129 (845-11/22/28)

Plants expressing PC TPS and Pat-TPS simultaneously were generated by retransformmg Pat-TPS lines (resistant against kanamycin) with construct pMOG1129, harbouring a PC TPS construct and a hygromycin resistance marker gene, resulting in genotypes pMOG1129 (845-11) , pMOG1129(845-22) and pMOG1129 (845-28) . Tuber-mass yield varied between almost no yield up to yield comparable or higher then control plants (Fig. 28) .

EXAMPLE 22: Tobacco

Performance of N. tabacum plants transgenic for TPS and TPP constructs

Root syεtem

Tobacco plants transgenic for 35S TPP (pMOGlOlO) or 35S TPS (pMOG799) were grown in the greenhouse. Root size was determined just before flowering. Lines transgenic for pMOGlOlO revealed a significantly smaller/larger root size compared to pMOG799 and non-transgemc wild- type tobacco plants.

Influence of expressing TPS and/or TPP on flowering

Tobacco plants transgenic for 35S-TPS, PC-TPS, 35S-TPP or PC-TPP were cultured in the greenhouse. Plants expressing high levels of the TPS gene revealed significantly slower growth rates compared to wild-type plants. Flowering and senescence of the lower leaves was delayed m these plants resulting in a stay-green phenotype of the normally senescing leaves. Plants expressing high levels of the TPP gene did not make any flowers or made aberrant, not fully developing flower buds resulting in sterility

Influence of expreεεing TPS and/or TPP on seed set ting

Tobacco plants transgenic for 35S-TPS, PC-TPS, 35S-TPP or PC-TPP were cultured in the greenhouse. Plants expressing high levels of the TPP gene revealed poor or no development of flowers and absence of seed- setting.

Influence of expreεsing TPS and/ or TPP on seed germination Tobacco plants transgenic for 35S TPP (pMOGlOlO) or PC TPP were grown in the greenhouse. Some of the transgenic lines, having low expression levels of the transgene, did flower and set seed. Upon germination of SI seed, a significantly reduced germination frequency was observed

(or germination was absent) compared to SI seed derived from wild-type plants (table 12). s

Table 12 . Germination of transgenic 35S-TPP seeds

Influence of expressing TPS and/or TPP on seed yield

Seed-yield was determined for SI plants transgenic for pMOG1010-5 On average, pMOG1010-5 yielded 4.9 g seed/ plant (n=8) compared to 7.8 g seed/ plant (n=8) for wild-type plants. The "1000-gram" weight is 0.06 g for line pMOG1010-5 compared to 0.08 g for wild-type Samsun NN. These data can be explained by a reduced export of carbohydrates from the source leaves, leading to poor development of seed "sink" tissue.

Influence of TPS and TPP expression on leaf morphology Segments of greenhouse grown PC-TPS transgenic, PC-TPP transgenic and non-transgenic control tobacco leaves were fixed, embedded m plastic and coupes were prepared to study cell structures using light- microscopy. Cell structures and morphology of cross-sections of the PC-TPP transgenic plants were comparable to those observed in control plants. Cross-sections of PC-TPS transgenics revealed that the spongy parenchyme cell-layer constituted of 7 layers of cells compared to 3 layers in wild-type and TPP transgenic plants (Fig. 29) This finding agrees with our observation that TPS transgenic plant lines form thicker and more rigid leaves compared to TPP and control plants

EXAMPLE 23 Inhibition of cold-sweetening by the expression of trehalose phosphate synthase Transgenic potato plants ( Solanum tuberosum cv Kardal) were generated harbouring the TPS gene under control of the potato tuber-specific patatm promoter (pMOG845; Example 1) . Transgenic plants and wild-type control plants were grown m the greenhouse and tubers were harvested. Samples of tuber material were taken for sugar analysis directly after harvesting and after 6 months of storage at 4°C. Data resulting from the HPLC-PED analysis are depicted in Fig. 30

What is clearly shown is that potato plants transgenic for TPS_{E coll} have a lower amount of total sugar (glucose, fructose and sucrose) accumulating m tubers directly after harvesting. After a storage period of 6 months at 4°C, the increase in soluble sugars is significantly less in the transgenic lines compared to the wild-type control lines. <7

EXAMPLE 24

Improved performance of 35S TPS 35S TPP (pMOG851) transgenic tobacco plants under drought stress

Transgenic tobacco plants were engineered harbouring both the TPS and TPP gene from E. coli under control of the 35S CaMV promoter. The expression of the TPS and TPP genes was verified in the lines obtained using Northern blot and enzyme activity measurements. pMOG851-2 was shown to accumulate 0.008 mg trehalose.g"¹ fw and pMOG851-5 accumulated 0.09 mg trehalose.g^_1 fw. Expression of both genes had a pronounced effect on plant morphology and growth performance under drought stress. When grown under drought stress imposed by limiting water supply, the two transgenic tobacco lines tested, pMOG851-2 and pMOG851-5, yielded total dry weights that were 28% (P<0.01) and 39% (P<0.001) higher than those of wild-type tobacco. These increaseε in dry weight were due mainly to mcreaεed leaf production: leaf dry weights were up to 85% higher for pMOG851-5 transgenic plants. No significant differences were observed under well-watered conditions.

Drough t stress experiments FI seeds obtained from self-fertilization of primary transformants pMOG851-2 and pMOG851-5 (Goddijn et al . (1997) Plant Physiol. 113, 181) were used in this study. Seeds were sterilized for 10 minutes m 20% household bleach, rinsed five times m sterile water, and sown on half-strength Murashige and Skoog medium containing 10 g.L^-1 sucrose and 100 mg.L'¹ kanamycin. Wildtype SRI seeds were sown on plates without kanamycin. After two weeks seedlings from all lines were transferred to soil (sandy loam) , and grown in a growth chamber at 22 °C at approximately 100 μE.irr² light intensity, 14h.d-^χ. All plants were grown in equal amounts of soil, in 3.8 liter pots. The plants were watered daily with half-strength Hoagland's nutrient solution.

The seedlings of pMOG851-2 and pMOG851-5 grew somewhat slower than the wildtype seedlings Since we considered it most important to start the experiments at equal developmental stage, we initiated the drought stress treatments of each line when the seedlings were at equal height (10 cm), at an equal developmental stage (4-leaves), and at equal dry weight (as measured from two additional plants of each line) . This meant that the onset of pMOG851-2 treatment was two days later than wildtype, and that of pMOG851-5 seven days later than wildtype. From each line, six plants were subjected to drought stress, while four were kept under well-watered conditions as controls. The wildtype tobacco plants were droughted by maintaining them around the wilting point: when the lower half of the leaves were wilted, the plants were given so much nutrient solution that the plants temporarily regained turgor. In practice, this meant supplying 50 ml of nutrient solution every three days; the control plants were watered daily to keep them at field capacity. The pMOG851-2 and pMOG851-5 plants were then watered in the exact same way as wildtype, i . e . , they were supplied with equal amounts of nutrient solution and after equal time intervals as wildtype. The stem height was measured regularly during the entire study period. All plants were harvested on the same day (32 d after the onset of treatment for the wildtype plants) , as harvesting the transgenic plants at a later stage would complicate the comparison of the plant lines. At the time of harvest the total leaf area was measured using a Delta-T Devices leaf area meter (Santa Clara, CA) . In addition, the fresh weight and dry weight of the leaves, stems and roots was determined.

A second experiment was done essentially in the same way, to analyze the osmotic potential of the plants. After 35 days of drought stress, samples from the youngest mature leaves were taken at the beginning of the light period (n=3) .

Air-drymg of detached l eaves

The water loss from air-dried detached leaves was measured from well-watered, four-week old pMOG851-2, pMOG851-5 and wildtype plants Per plant line, five plants were used, and from each plant the two youngest mature leaves were detached and airdried at 25% relative humidity. The fresh weight of each leaf was measured over 32 hours. At the time of the experiment samples were taken from comparable, well-watered leaves, for osmotic potential measurements and determination of soluble sugar contents.

Osmoti c potenti al measurements

Leaf samples for osmotic potential analysis were immediately stored in capped 1 ml syringes and frozen on dry ice. Just before analysis the leaf sap was squeezed mto a small vial, mixed, and used to saturate a paper disc. The osmotic potential was then determined in Wescor C52 chambers, usmg a Wescor HR-33T dew point microvolt meter. <•>

Chlorophyl l fluorescence

Chlorophyll fluorescence of the wildtype, pMOG851-2 and pMOG851-5 plants was measured for each plant line after 20 days of drought treatment, using a pulse modulation (PAM) fluorometer (Walz, Effeltrich, Germany) Before the measurements, the plants were kept in the dark for two hours, followed by a one-hour light period Subsequently, the youngest mature leaf was dark-adapted for 20 minutes. At the beginning of each measurement, a small (0.05 μmol rrr² s"¹ modulated at 1.6 KHz) measuring light beam was turned on, and the minimal fluoreεcence level (FQ) was meaεured The maximal fluorescence level (F_m) was then measured by applying a saturation light pulse of 4000 μmol rrr² s^_1, 800 ms in duration After another 20 s, when the signal was relaxed to near F₀, brief saturating pulses of actinic light (800 ms in length, 4000 μmol m ² s^-1) were given repetitively for 30 s with 2 s dark intervals The photochemical (q_Q) and non- photochemical (q_E) quenching components were determined from the fluorescence/time curve according to Bolhar-Nordenkampf and Oquist (1993) At the moment of measurement, the leaves in question were not visibly wilted Statistical data were obtained by one-way analysis of variance using the program Number Cruncher Statistical System (Dr J.L Hmtze, 865 East 400 North, Kaysville, UT 84037, USA).

Chlorophyll fluorescence analysis of drought-stressed plants showed a higher photochemical quenching (qQ) and a higher ratio of variable fluorescence over maximal fluorescence (F_v/F_m) in pMOG851-5, indicating a more efficiently working photosynthetic machinery (table 13) .

/ P 7/02497

Table 13. Chlorophyll fluorescence parameters of wild-type (wt) and trehalose-accumulating (pMOG851-2, pMOG851-5) transgenic tobacco plants. P (probability) values were obtained from ANOVA tests analyzing differences per plant line between plants grown under well-watered (control) or dry conditions, as well as differences between each of the transgenic lines and WT, grown under well-watered or dry conditions. F_m: maximal fluorescence; F_v: variable fluorescence (F_m-Fo) : qς: photochemical quenching: q_E: non- photochemical quenching. F_m, F_v are expressed in arbitrary units (chart mm) .

7'

Carbohydrate analysis

At the time of harvest, pMOG851-5 plants contained 0.2 mg.g-¹ dry weight trehalose, whereas in pMOG851-2 and wildtype the trehalose levels were below the detection limit, under both stressed and unstressed conditions. The trehalose content in pMOG851-5 plants was comparable in stressed and unstressed plants (0.19 and 0.20 mg. g^_1 dry weight, respectively) . Under well-watered conditions, the levelε of glucose and fructose were twofold higher in pMOG851-5 plants than in wildtype. Leaves of stressed pMOG851-5 plants contained about threefold higher levels of each of the four nonstructural carbohydrates starch, sucrose, glucose and fructose, than leaves of stressed wildtype plants. In pMOG851-2 leaves, carbohydrate levels, like chlorophyll fluorescence values, did not differ significantly from those in wildtype. Stresεed plants of all lines contained increased levels of glucose and fructose compared to unstressed plants.

Osmotic potential of drought stressed and control plants

During a second, similar experiment under greenhouse conditions, the transgenic plants showed the same phenotypes as described above, and again the pMOG851-5 plants showed much less reduction in growth under drought stress than pMOG851-2 and wildtype plants. The osmotic potential m leaves of droughted pMOG851-5 plants (-1.77 ± 0.39 Mpa) was significantly lower (P=0.017) than in wildtype leaves (-1.00 ± 0.08 Mpa); pMOG851-2 showed intermediate values (-1.12 +, 0.05 Mpa). Similarly, under well-watered conditions the osmotic potential of PMOG851-5 plants (-0.79 ± 0.05 Mpa) was significantly lower (P=0.038) than that of wildtype leaves (-0.62 +. 0.03 Mpa), with pMOG851-2 having intermediate values (-0.70 ± 0.01 Mpa).

Airdrvmα of detached leaves

Leaves of pMOG851-2, pMOG851-5 and wildtype were detached and their fresh weight was measured over 32 hours of airdrying. Leaves of PMOG851-2 and pMOG851-5 plants lost significantly less water (P<0.05) than wildtype leaves: after 32 h leaves of pMOG851-5 and pMOG851-2 had 44% and 41% of their fresh weight left, respectively, compared to 30% for wildtype. At the time of the experiment samples were taken from comparable, well-watered leaves for osmotic potential determination and analysis of trehalose, sucrose, glucose and fructose. The two 7* transgenic lines had lower osmotic potentials than wildtype (P< 0.05), with pMOG851-5 having the lowest water potential (-0.63 ± 0.03 Mpa) , wildtype the highest (-0.51 + 0 02 Mpa) and pMOG851-2 intermediate (-0.57 ± 0.04 Mpa) . The levels of all sugars tested were significantly higher in leaves of pMOG851-5 plants than for wildtype leaves resulting m a threefold higher level of the four sugars combined (P = 0.002) . pMOG851-2 plants contained twofold higher levels of the four sugars combined (P = 0.09) . The trehalose levels were 0.24 +_ 0.02 mg.g^-1 DW in pMOG851-5 plants, and below detection in pMOG851-2 and wildtype.

EXAMPLE 25 Performance of TPS and TPP transgenic lettuce plant lines under drought stress Primary TPS and TPP transformants and wild-type control plants were subjected to drought-stress. Lines transgenic for TPP reached their wilting point first, then control plants, followed by TPS transgenic plants indicating that TPS transgenic lines, as observed in other plant species, have a clear advantage over the TPP and wild-type plants during drought stress.

EXAMPLE 26 Bolting of lettuce plants is affected in plants transgenic for PC-TPS or PC-TPP Bolting of lettuce is reduced in plants transgenic for PC-TPP (table 14). Plant lines transgenic for PC-TPS εhow enhanced bolting compared to wild-type lettuce plants.

Table 14 Bolting of lettuce plants

EXAMPLE 27

Performance of tomato plants transgenic for TPS and TPP

Constructs used in tomato transformation experiments: 35S TPP, PC-TPS, PC-TPS as-trehalase, PC-TPP, E8-TPS, E8-TPP, E8 TPS E8 as-trehalase. Plants transgenic for the TPP gene driven by the plastocyanm promoter and 35S promoter revealed phenotypes similar to those observed in other plants: bleaching of leaves, reduced formation of flowers or absent flower formation leading to small fruits or absence of fruits A small number of 35S-TPP transgenic lines generated extreme large fruits. Those fruits revealed enhanced outgrow of the pericarp. Plants transgenic for the TPS gene driven by the plastocyanm promoter and 35S promoter did not form small lancet shaped leaves. Some severely stunted plants did form small dark-green leaves. Plants transgenic for PC-TPS and PC-as-trehalase did form smaller and darker green leaves as compared to control plants. The colour and leaf-edge of the 35S or PC driven TPS and TPP transgenic plants were clearly distinguishable similar to what is observed in other crops.

Plants harbouring the TPS and TPP gene under control of the fruit- 7Λ specific E8 promoter did not show any phenotypical differences compared to wild-type fruits Plants transgenic for E8 TPS E8 as- trehalase produced aberrant fruits with a yellow skin and incomplete ripening.

EXAMPLE 28 Performance of potato plants transgenic for as-trehalase and/or TPS

Constructs: 35S as-trehalase (pMOG1027) and 35S as-trehalase Pat TPS (PMOG1027 (845-11/22/28)

Plants expressing 35S as-trehalase and pat-TPS simultaneously were generated by retransformmg pat-TPS lines (resistant agamst kanamycin) with construct pMOG1027, harbouring the 35S as-trehalaεe construct and a hygromycin resistance marker gene, resultmg in genotypes pMOG1027(845-11) , pMOG1027 (845-22) and pMOG1027 (845-28) . Microtubers were induced in vi tro and fresh weight of the microtubers was determined. The average fresh weight yield was increased for transgenic lines harbouring pMOG1027 (pMOG845-ll/22/28) . The fresh weight biomass of microtubers obtained from lines transgenic for pMOG1027 only was slightly higher then wild-type control plants. Resulting plants were grown in the greenhouse and tuber yield was determined (Fig. 33). Lines transgenic for 35S as-trehalase or a combination of 35S as-trehalase and pat-TPS yielded significantly more tuber-mass compared to control lines. Starch determination revealed no difference in starch content of tubers produced by plant lines having a higher yield (Fig. 34) A large number of the 1027(845-11/22/28) lines produced tubers above the soil out of the axillary buds of the leaves indicating a profound influence of the constructs used on plant development. Plant lines transgenic for 35S as-trehalase only did not form tubers above the soil.

Constructs: Pat as-trehalase (pMOG1028) and Pat as-trehalase Pat TPS (pMOG1028(845-ll/22/28) ) Plants expressing Pat as-trehalase and Pat-TPS simultaneously were generated by retransforming Pat-TPS lines (resistant agamst kanamycin) with construct pMOG1028, harbouring the Pat as-trehalase construct and a hygromycin resistance marker gene, resulting in genotypes pMOG1028 (845-11) , pMOG1028 (845-22) and pMOG1028 (845-28) . Plants were grown in the greenhouse and tuber yield was determined (Fig. 35) . A number of pMOG1028 transgenic lines yielded significantly more tuber-mass compared to control lines Individual plants transgenic for both Pat TPS and Pat as-trehalase revealed a varying tuber-yield from almost no yield up to a yield comparable to or higher then the control-lines (Fig 35)

Construct: PC as-trehalase (pMOG1092)

Plants transgenic for pMOG1092 were grown m the greenhouse and tuber- yield was determined Several lines formed darker-green leaves compared to controls Tuber-yield was significantly enhanced compared to non-transgenic plants (Fig. 36).

Construct. PC as-trehalase PC-TPS (pMOG 1130) Plants transgenic for pMOG 1130 were grown in the greenhouse and tuber-yield was determined Several transgenic lines developed small dark-green leaves and severely stunted growth indicating that the phenotypic effects observed when plants are transformed with TPS is more severe when the as-trehalase gene is expressed simultaneously (see Example 21) Tuber-mass yield varied between almost no yield up to significantly more yield compared to control plants (Fig 37)

EXAMPLE 29 Overexpression of a potato trehalase cDNA in N. tabacum Construct de35S CaMV trehalase (pMOG1078)

Primary tooacco transformants transgenic for pMOGl078 revealed a phenotype different from wild-type tobacco, some transgenics have a dark-green leaf colour and a thicker leaf (the morphology of the leaf is not lancet-shaped) indicating an influence of trehalase gene- expression on plant metabolism. Seeds of selfed primary transformants were sown and selected on kanamycin. The phenotype showed to segregate in a mendelian fashion in the SI generation. DEP OS I T S

The following deposits were made under the Budapest Treaty The clones were deposited at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, P.O Box 273, 3740 AG Baarn, The Netherlands on April 21, 1997 and received the following numbers:

Escherichia coli DH5alpha/pMOG1192 CBS 692.97 DH5alpha/pMOG1240 CBS 693.97 DH5alpha/pMOG1241 CBS 694.97 DH5alpha/pMOGl242 CBS 695.97 DH5alpha/pMOG1243 CBS 696.97 DH5alpha/pMOG1244 CBS 697.97 DH5alpha/pMOG1245 CBS 698 97

Deposited clones

PMOG1192 harbors the Hel ianthus annuus TPS/TPP bipartite cDNA inserted in the multi-copy vector pGEM-T (Promega) . pMOG1240 harbors the tobacco TPS "825" bp cDNA fragment inserted in pCRscript (Stratagene) . pMOG1241 harbors the tobacco TPS "840" bp cDNA fragment inserted in pGEM-T (Promega) pMOG1242 harbors the tobacco TPS "630" bp cDNA fragment inserted m pGEM-T (Promega) . pMOG1243 harbors the tobacco TPP "543" bp cDNA fragment inserted m pGEM-T (Promega) PMOG1244 harbors the tobacco TPP "723" bp cDNA fragment inserted in a pUC18 plasmid. PMOG1245 harbors the tobacco TPP "447" bp fragment inserted in pGEM-T (Promega) .

List of relevant pMQG### and pVDH### clones

1. Binary vectors pMOG23 Binary vector (ca. 10 Kb) harboring the NPTII selection marker pMOG22 Derivative of pMOG23, the NPTII-gene has been replaced by the HPT-gene which confers resistance to hygromycme pVDH 275 Binary vector derived from pMOG23, harbors a plastocyanm promoter- nos terminator expression cassette. pMOG402 Derivative of pMOG23, a pomt-mutation m the NPTII-gene has been restored, no Kpnl restriction site present in the polylinker pMOG800 Derivative of pMOG402 with restored Kpnl site in polylinker

2. TPS / TPP expression constructs pMOG 799 35S-TPS-3'nosl pMOG 810 idem with Hyg marker pMOG 845 Pat-TPS-3 'PotPiII pMOG 925 idem with Hyg marker pMOG 851 35S-TPS-3'nos 35S-TPP(atg)² pMOG 1010 de35S CaMV amv leader TPP(gtg) PotPiII pMOG 1142 idem with Hyg marker pMOG 1093 Plastocyanm- TPS-3'nos pMOG 1129 idem with Hyg marker pMOG 1177 Plastocyanm- TPS-3 'PotPiII 3 'nos pVDH 318 Identical to pMOG1177 Functionally identical to pMOG1093 pMOG 1124 Plastocyanm- TPP(gtg) 3'PotPιII 3'nos pVDH 321 Identical to pMOG1124 pMOG 1128 Patatm TPP(gtg) 3 ' PotPiII pMOG 1140 E8-TPS-3 'nos pMOG 1141 E8-TPP(gtg) -3 PotPiII

3. Trehalase constructs pMOG 1028 Patatin as-trehalase 3 'PotPiII, Hygromycin resistance marker pMOG 1078 de35S CaMV amv leader trehalase 3'nos pMOG 1090 de35S CaMV amv leader as-trehalase 3'nos pMOG 1027 idem with Hyg marker pMOG 1092 Plastocyanm- as trehalase-3 'nos pMOG 1130 Plastocyanm- as trehalase-3 'nos Plastocyanm-TPS-3 'nos pMOG 1153 E8-TPS-3'noε E8-as trehalase-3 ' PotPiII

All constructs harbour the NPTII selection marker unless noted otherwise

Two types of TPP constructs have been used as described in

Goddijn et al . (1997) Plant Physiol.113, 181. ₇&

SEQUENCE LISTING

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(C) CITY: Leiden (E) COUNTRY: The Netherlands

(F) POSTAL CODE (ZIP) : 2333 CB

(G) TELEPHONE: (0)71-5258282 (H) TELEFAX: (0)71-5221471 (ii) TITLE OF INVENTION: Regulating metabolism by modifying the level of trehalose-6-phoεphate

(iii) NUMBER OF SEQUENCES: 57 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: EP 96.201.225.8

(B) FILING DATE: 03-MAY-1996 (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: EP 96.202.128.3

(B) FILING DATE: 26-JUL-1996

(vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: EP 96.202.395.8

(B) FILING DATE: 29-AUG-1996

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1450 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 21..1450 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 7°)

ATAAAACTCT CCCCGGGACC ATG ACT ATG AGT CGT TTA GTC GTA GTA TCT 50

Met Thr Met Ser Arg Leu Val Val Val Ser

1 5 10 AAC CGG ATT GCA CCA CCA GAC GAG CAC GCC GCC AGT GCC GGT GGC CTT 98 Asn Arg He Ala Pro Pro Asp Glu His Ala Ala Ser Ala Gly Gly Leu 15 20 25

GCC GTT GGC ATA CTG GGG GCA CTG AAA GCC GCA GGC GGA CTG TGG TTT 146 Ala Val Gly He Leu Gly Ala Leu Lys Ala Ala Gly Gly Leu Trp Phe 30 35 40

GGC TGG AGT GGT GAA ACA GGG AAT GAG GAT CAG CCG CTA AAA AAG GTG 194 Gly Trp Ser Gly Glu Thr Gly Asn Glu Asp Gin Pro Leu Lys Lys Val 45 50 55

AAA AAA GGT AAC ATT ACG TGG GCC TCT TTT AAC CTC AGC GAA CAG GAC 242

Lys Lys Gly Asn He Thr Trp Ala Ser Phe Asn Leu Ser Glu Gin Asp

60 65 70

CTT GAC GAA TAC TAC AAC CAA TTC TCC AAT GCC GTT CTC TGG CCC GCT 290

Leu Asp Glu Tyr Tyr Asn Gin Phe Ser Asn Ala Val Leu Trp Pro Ala

75 80 85 90 TTT CAT TAT CGG CTC GAT CTG GTG CAA TTT CAG CGT CCT GCC TGG GAC 338

Phe His Tyr Arg Leu Asp Leu Val Gin Phe Gin Arg Pro Ala Trp Asp 95 100 105

GGC TAT CTA CGC GTA AAT GCG TTG CTG GCA GAT AAA TTA CTG CCG CTG 386 Gly Tyr Leu Arg Val Asn Ala Leu Leu Ala Asp Lys Leu Leu Pro Leu

110 115 120

TTG CAA GAC GAT GAC ATT ATC TGG ATC CAC GAT TAT CAC CTG TTG CCA 434 Leu Gin Asp Asp Asp He He Trp He His Asp Tyr His Leu Leu Pro 125 130 135

TTT GCG CAT GAA TTA CGC AAA CGG GGA GTG AAT AAT CGC ATT GGT TTC 482

Phe Ala His Glu Leu Arg Lys Arg Gly Val Asn Asn Arg He Gly Phe

140 145 150

TTT CTG CAT ATT CCT TTC CCG ACA CCG GAA ATC TTC AAC GCG CTG CCG 530

Phe Leu His He Pro Phe Pro Thr Pro Glu He Phe Asn Ala Leu Pro

155 160 165 170 ACA TAT GAC ACC TTG CTT GAA CAG CTT TGT GAT TAT GAT TTG CTG GGT 578

Thr Tyr Asp Thr Leu Leu Glu Gin Leu Cys Asp Tyr Asp Leu Leu Gly 175 180 185

TTC CAG ACA GAA AAC GAT CGT CTG GCG TTC CTG GAT TGT CTT TCT AAC 626 Phe Gin Thr Glu Asn Asp Arg Leu Ala Phe Leu Asp Cys Leu Ser Asn

190 195 200

CTG ACC CGC GTC ACG ACA CGT AGC GCA AAA AGC CAT ACA GCC TGG GGC 674 Leu Thr Arg Val Thr Thr Arg Ser Ala Lys Ser His Thr Ala Trp Gly 205 210 215 S>o

AAA GCA TTT CGA ACA GAA GTC TAC CCG ATC GGC ATT GAA CCG AAA GAA 722

Lyε Ala Phe Arg Thr Glu Val Tyr Pro He Gly He Glu Pro Lys Glu

220 225 230 ATA GCC AAA CAG GCT GCC GGG CCA CTG CCG CCA AAA CTG GCG CAA CTT 770

He Ala Lys Gin Ala Ala Gly Pro Leu Pro Pro Lys Leu Ala Gin Leu

235 240 245 250

AAA GCG GAA CTG AAA AAC GTA CAA AAT ATC TTT TCT GTC GAA CGG CTG 818 Lys Ala Glu Leu Lys Asn Val Gin Asn He Phe Ser Val Glu Arg Leu

255 260 265

GAT TAT TCC AAA GGT TTG CCA GAG CGT TTT CTC GCC TAT GAA GCG TTG 866

Asp Tyr Ser Lys Gly Leu Pro Glu Arg Phe Leu Ala Tyr Glu Ala Leu 270 275 280

CTG GAA AAA TAT CCG CAG CAT CAT GGT AAA ATT CGT TAT ACC CAG ATT 914

Leu Glu Lys Tyr Pro Gin His His Gly Lys He Arg Tyr Thr Gin He

285 290 295

GCA CCA ACG TCG CGT GGT GAT GTG CAA GCC TAT CAG GAT ATT CGT CAT 962

Ala Pro Thr Ser Arg Gly Asp Val Gin Ala Tyr Gin Asp He Arg His

300 305 310 CAG CTC GAA AAT GAA GCT GGA CGA ATT AAT GGT AAA TAC GGG CAA TTA 1010

Gin Leu Glu Asn Glu Ala Gly Arg He Asn Gly Lys Tyr Gly Gin Leu

315 320 325 330

GGC TGG ACG CCG CTT TAT TAT TTG AAT CAG CAT TTT GAC CGT AAA TTA 1058 Gly Trp Thr Pro Leu Tyr Tyr Leu Asn Gin His Phe Asp Arg Lys Leu

335 340 345

CTG ATG AAA ATA TTC CGC TAC TCT GAC GTG GGC TTA GTG ACG CCA CTG 1106

Leu Met Lys He Phe Arg Tyr Ser Asp Val Gly Leu Val Thr Pro Leu 350 355 360

CGT GAC GGG ATG AAC CTG GTA GCA AAA GAG TAT GTT GCT GCT CAG GAC 1154

Arg Asp Gly Met Asn Leu Val Ala Lys Glu Tyr Val Ala Ala Gin Asp

365 370 375

CCA GCC AAT CCG GGC GTT CTT GTT CTT TCG CAA TTT GCG GGA GCG GCA 1202

Pro Ala Asn Pro Gly Val Leu Val Leu Ser Gin Phe Ala Gly Ala Ala

380 385 390 AAC GAG TTA ACG TCG GCG TTA ATT GTT AAC CCC TAC GAT CGT GAC GAA 1250

Asn Glu Leu Thr Ser Ala Leu He Val Asn Pro Tyr Asp Arg Asp Glu

395 400 405 410

GTT GCA GCT GCG CTG GAT CGT GCA TTG ACT ATG TCG CTG GCG GAA CGT 1298 Val Ala Ala Ala Leu Asp Arg Ala Leu Thr Met Ser Leu Ala Glu Arg

415 420 425

ATT TCC CGT CAT GCA GAA ATG CTG GAC GTT ATC GTG AAA AAC GAT ATT 1346

He Ser Arg His Ala Glu Met Leu Asp Val He Val Lys Asn Asp He 430 435 440 ε>ι

AAC CAC TGG CAG GAG TGC TTC ATT AGC GAC CTA AAG CAG ATA GTT CCG 1394 Asn His Trp Gin Glu Cys Phe He Ser Asp Leu Lys Gin He Val Pro 445 450 455 CGA AGC GCG GAA AGC CAG CAG CGC GAT AAA GTT GCT ACC TTT CCA AAG 1442 Arg Ser Ala Glu Ser Gin Gin Arg Asp Lys Val Ala Thr Phe Pro Lys 460 465 470

CTC TGC AG 1450 Leu Cys 475

(2) INFORMATION FOR SEQ ID NO: 2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 476 ammo acids

(B) TYPE: ammo acid (D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Thr Met Ser Arg Leu Val Val Val Ser Asn Arg He Ala Pro Pro 1 5 10 15

Asp Glu His Ala Ala Ser Ala Gly Gly Leu Ala Val Gly He Leu Gly 20 25 30

Ala Leu Lys Ala Ala Gly Gly Leu Trp Phe Gly Trp Ser Gly Glu Thr 35 40 45

Gly Asn Glu Asp Gin Pro Leu Lys Lys Val Lys Lys Gly Asn He Thr 50 55 60

Trp Ala Ser Phe Asn Leu Ser Glu Gin Asp Leu Asp Glu Tyr Tyr Asn 65 70 75 80 Gin Phe Ser Asn Ala Val Leu Trp Pro Ala Phe His Tyr Arg Leu Asp

85 90 95

Leu Val Gin Phe Gin Arg Pro Ala Trp Asp Gly Tyr Leu Arg Val Asn 100 105 110

Ala Leu Leu Ala Asp Lys Leu Leu Pro Leu Leu Gin Asp Asp Asp He 115 120 125

He Trp He His Asp Tyr His Leu Leu Pro Phe Ala His Glu Leu Arg 130 135 140

Lys Arg Gly Val Asn Asn Arg He Gly Phe Phe Leu His He Pro Phe 145 150 155 160 Pro Thr Pro Glu He Phe Asn Ala Leu Pro Thr Tyr Asp Thr Leu Leu

165 170 175 Glu Gin Leu Cys Asp Tyr Asp Leu Leu Gly Phe Gin Thr Glu Asn Asp 180 185 190

Arg Leu Ala Phe Leu Asp Cys Leu Ser Asn Leu Thr Arg Val Thr Thr 195 200 205

Arg Ser Ala Lys Ser His Thr Ala Trp Gly Lys Ala Phe Arg Thr Glu 210 215 220 Val Tyr Pro He Gly He Glu Pro Lys Glu He Ala Lys Gin Ala Ala 225 230 235 240

Gly Pro Leu Pro Pro Lys Leu Ala Gin Leu Lys Ala Glu Leu Lys Asn 245 250 255

Val Gin Asn He Phe Ser Val Glu Arg Leu Asp Tyr Ser Lys Gly Leu 260 265 270

Pro Glu Arg Phe Leu Ala Tyr Glu Ala Leu Leu Glu Lys Tyr Pro Gin 275 280 285

His His Gly Lys He Arg Tyr Thr Gin He Ala Pro Thr Ser Arg Gly 290 295 300 Asp Val Gin Ala Tyr Gin Asp He Arg His Gin Leu Glu Asn Glu Ala 305 310 315 320

Gly Arg He Asn Gly Lys Tyr Gly Gin Leu Gly Trp Thr Pro Leu Tyr 325 330 335

Tyr Leu Asn Gin His Phe Asp Arg Lys Leu Leu Met Lys He Phe Arg 340 345 350

Tyr Ser Asp Val Gly Leu Val Thr Pro Leu Arg Asp Gly Met Asn Leu 355 360 365

Val Ala Lys Glu Tyr Val Ala Ala Gin Asp Pro Ala Asn Pro Gly Val 370 375 380 Leu Val Leu Ser Gin Phe Ala Gly Ala Ala Asn Glu Leu Thr Ser Ala 385 390 395 400

Leu He Val Asn Pro Tyr Asp Arg Asp Glu Val Ala Ala Ala Leu Asp 405 410 415

Arg Ala Leu Thr Met Ser Leu Ala Glu Arg He Ser Arg His Ala Glu 420 425 430

Met Leu Asp Val He Val Lys Asn Asp He Asn His Trp Gin Glu Cys 435 440 445

Phe He Ser Asp Leu Lys Gin He Val Pro Arg Ser Ala Glu Ser Gin 450 455 460 Gin Arg Asp Lys Val Ala Thr Phe Pro Lys Leu Cys 465 470 475 (2) INFORMATION FOR SEQ ID NO: 3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 835 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (ill) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION. 18..818

(XI) SEQUENCE DESCRIPTION. SEQ ID NO: 3:

ATAAAACTCT CCCCGGG ATG ACA GAA CCG TTA ACC GAA ACC CCT GAA CTA 50 Met Thr Glu Pro Leu Thr Glu Thr Pro Glu Leu

1 5 10

TCC GCG AAA TAT GCC TGG TTT TTT GAT CTT GAT GGA ACG CTG GCG GAA 98 Ser Ala Lys Tyr Ala Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu 15 20 25

ATC AAA CCG CAT CCC GAT CAG GTC GTC GTG CCT GAC AAT ATT CTG CAA 146

He Lys Pro His Pro Asp Gin Val Val Val Pro Asp Asn He Leu Gin

30 35 40

GGA CTA CAG CTA CTG GCA ACC GCA AGT GAT GGT GCA TTG GCA TTG ATA 194

Gly Leu Gin Leu Leu Ala Thr Ala Ser Asp Gly Ala Leu Ala Leu He

45 50 55 TCA GGG CGC TCA ATG GTG GAG CTT GAC GCA CTG GCA AAA CCT TAT CGC 242 Ser Gly Arg Ser Met Val Glu Leu Asp Ala Leu Ala Lys Pro Tyr Arg 60 65 70 75

TTC CCG TTA GCG GGC GTG CAT GGG GCG GAG CGC CGT GAC ATC AAT GGT 290 Phe Pro Leu Ala Gly Val His Gly Ala Glu Arg Arg Asp He Asn Gly

80 85 90

AAA ACA CAT ATC GTT CAT CTG CCG GAT GCG ATT GCG CGT GAT ATT AGC 338 Lys Thr His He Val His Leu Pro Asp Ala He Ala Arg Asp He Ser 95 100 105

GTG CAA CTG CAT ACA GTC ATC GCT CAG TAT CCC GGC GCG GAG CTG GAG 386 Val Gin Leu His Thr Val He Ala Gin Tyr Pro Gly Ala Glu Leu Glu 110 115 120

GCG AAA GGG ATG GCT TTT GCG CTG CAT TAT CGT CAG GCT CCG CAG CAT 434 Ala Lys Gly Met Ala Phe Ala Leu His Tyr Arg Gin Ala Pro Gin His 125 130 135 9+

GAA GAC GCA TTA ATG ACA TTA GCG CAA CGT ATT ACT CAG ATC TGG CCA 482 Glu Asp Ala Leu Met Thr Leu Ala Gin Arg He Thr Gin He Trp Pro 140 145 150 155 CAA ATG GCG TTA CAG CAG GGA AAG TGT GTT GTC GAG ATC AAA CCG AGA 530 Gin Met Ala Leu Gin Gin Gly Lys Cys Val Val Glu He Lys Pro Arg 160 165 170

GGT ACC AGT AAA GGT GAG GCA ATT GCA GCT TTT ATG CAG GAA GCT CCC 578 Gly Thr Ser Lys Gly Glu Ala He Ala Ala Phe Met Gin Glu Ala Pro 175 180 185

TTT ATC GGG CGA ACG CCC GTA TTT CTG GGC GAT GAT TTA ACC GAT GAA 626 Phe He Gly Arg Thr Pro Val Phe Leu Gly Asp Asp Leu Thr Asp Glu 190 195 200

TCT GGC TTC GCA GTC GTT AAC CGA CTG GGC GGA ATG TCA GTA AAA ATT 674 Ser Gly Phe Ala Val Val Asn Arg Leu Gly Gly Met Ser Val Lys He 205 210 215

GGC ACA GGT GCA ACT CAG GCA TCA TGG CGA CTG GCG GGT GTG CCG GAT 722 Gly Thr Gly Ala Thr Gin Ala Ser Trp Arg Leu Ala Gly Val Pro Asp 220 225 230 235 GTC TGG AGC TGG CTT GAA ATG ATA ACC ACC GCA TTA CAA CAA AAA AGA 770

Val Trp Ser Trp Leu Glu Met He Thr Thr Ala Leu Gin Gin Lys Arg 240 245 250

GAA AAT AAC AGG AGT GAT GAC TAT GAG TCG TTT AGT CGT AGT ATC TAA 818 Glu Asn Asn Arg Ser Asp Asp Tyr Glu Ser Phe Ser Arg Ser He *

255 260 265

CCGGATTGCA CCTGCAG 835

270

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 272 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Thr Glu Pro Leu Thr Glu Thr Pro Glu Leu Ser Ala Lys Tyr Ala 1 5 10 15

Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu He Lys Pro His Pro 20 25 30 S^>5

Asp Gin Val Val Val Pro Asp Asn He Leu Gin Gly Leu Gin Leu Leu 35 40 45

Ala Thr Ala Ser Asp Gly Ala Leu Ala Leu He Ser Gly Arg Ser Met 50 55 60

Val Glu Leu Asp Ala Leu Ala Lys Pro Tyr Arg Phe Pro Leu Ala Gly 65 70 75 80 Val His Gly Ala Glu Arg Arg Asp He Asn Gly Lys Thr His He Val

85 90 95

His Leu Pro Asp Ala He Ala Arg Asp He Ser Val Gin Leu His Thr 100 105 110

Val He Ala Gin Tyr Pro Gly Ala Glu Leu Glu Ala Lys Gly Met Ala 115 120 125

Phe Ala Leu His Tyr Arg Gin Ala Pro Gin His Glu Asp Ala Leu Met 130 135 140

Thr Leu Ala Gin Arg He Thr Gin He Trp Pro Gin Met Ala Leu Gin 145 150 155 160 Gin Gly Lys Cys Val Val Glu He Lys Pro Arg Gly Thr Ser Lys Gly

165 170 175

Glu Ala He Ala Ala Phe Met Gin Glu Ala Pro Phe He Gly Arg Thr 180 185 190

Pro Val Phe Leu Gly Asp Asp Leu Thr Asp Glu Ser Gly Phe Ala Val 195 200 205

Val Asn Arg Leu Gly Gly Met Ser Val Lys He Gly Thr Gly Ala Thr 210 215 220

Gin Ala Ser Trp Arg Leu Ala Gly Val Pro Asp Val Trp Ser Trp Leu 225 230 235 240 Glu Met He Thr Thr Ala Leu Gin Gin Lys Arg Glu Asn Asn Arg Ser

245 250 255

Asp Asp Tyr Glu Ser Phe Ser Arg Ser He * 260 265

(2) INFORMATION FOR SEQ ID NO: 5:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AAGCTTATGT TGCCATATAG AGTAGAT 27

(2) INFORMATION FOR SEQ ID NO: 6:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(m) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GTAGTTGCCA TGGTGCAAAT GTTCATATG 29

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAYITIATIT GGRTICAYGA YTAYCA 26

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: lmear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TIGGITKITT YYTICAYAYI CCITTYCC 28

(2) INFORMATION FOR SEQ ID NO: 9: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (in) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GYIACIARRT TCATICCRTC IC 22

!2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 743 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (ill) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1..743

(D) OTHER INFORMATION: /partial

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GAC GTG ATG TGG ATG CAC GAC TAC CAT TTG ATG GTG TTG CCT ACG TTC 48 Asp Val Met Trp Met His Asp Tyr His Leu Met Val Leu Pro Thr Phe 1 5 10 15 TTG AGG AGG CGG TTC AAT CGT TTG AGA ATG GGG TTT TTC CTT CAC AGT 96 Leu Arg Arg Arg Phe Asn Arg Leu Arg Met Gly Phe Phe Leu His Ser 20 25 30

CCA TTT CCC TCA TCT GAG ATT TAC AGG ACA CTT CCT GTT AGA GAG GAA 144 Pro Phe Pro Ser Ser Glu He Tyr Arg Thr Leu Pro Val Arg Glu Glu 35 40 45 P 7

8©

ATA CTC AAG GCT TTG CTC TGT GCT GAC ATT GTT GGA TTC CAC ACT TTT 192 He Leu Lys Ala Leu Leu Cys Ala Asp He Val Gly Phe His Thr Phe 50 55 60 GAC TAC GCG AGA CAC TTC CTC TCT TGT TGC AGT CGG ATG TTG GGT TTA 240 Asp Tyr Ala Arg His Phe Leu Ser Cys Cys Ser Arg Met Leu Gly Leu 65 70 75 80

GAG TAT CAG TCT AAA AGA GGT TAT ATA GGG TTA GAA TAC TAT GGA CGG 288 Glu Tyr Gin Ser Lys Arg Gly Tyr He Gly Leu Glu Tyr Tyr Gly Arg

85 90 95

ACA GTA GGC ATC AAG ATT ATG CCC GTC GGG ATA CAT ATG GGT CAT ATT 336 Thr Val Gly He Lys He Met Pro Val Gly He His Met Gly His He 100 105 HO

GAG TCC ATG AAG AAA CTT GCA GCG AAA GAG TTG ATG CTT AAG GCG CTA 384

Glu Ser Met Lys Lys Leu Ala Ala Lys Glu Leu Met Leu Lys Ala Leu

115 120 125

AAG CAG CAA TTT GAA GGG AAA ACT GTG TTG CTT GGT GCC GAT GAC CTG 432

Lys Gin Gin Phe Glu Gly Lys Thr Val Leu Leu Gly Ala Asp Asp Leu 130 135 140 GAT ATT TTC AAA GGT ATA AAC TTA AAG CTT CTA GCT ATG GAA CAG ATG 480 Asp He Phe Lys Gly He Asn Leu Lys Leu Leu Ala Met Glu Gin Met 145 150 155 160

CTC AAA CAG CAC CCC AAG TGG CAA GGG CAG GCT GTG TTG GTC CAG ATT 528 Leu Lys Gin His Pro Lys Trp Gin Gly Gin Ala Val Leu Val Gin He

165 170 175

GCA AAT CCT ACG AGG GGT AAA GGA GTA GAT TTT GAG GAA ATA CAG GCT 576 Ala Asn Pro Thr Arg Gly Lys Gly Val Asp Phe Glu Glu He Gin Ala 180 185 190

GAG ATA TCG GAA AGC TGT AAG AGA ATC AAT AAG CAA TTC GGC AAG CCT 624

Glu He Ser Glu Ser Cys Lys Arg He Asn Lys Gin Phe Gly Lys Pro

195 200 205

GGA TAT GAG CCT ATA GTT TAT ATT GAT AGG CCC GTG TCA AGC AGT GAA 672

Gly Tyr Glu Pro He Val Tyr He Asp Arg Pro Val Ser Ser Ser Glu

210 215 220 CGC ATG GCA TAT TAC AGT ATT GCA GAA TGT GTT GTT GTC ACG GCT GTG 720 Arg Met Ala Tyr Tyr Ser He Ala Glu Cys Val Val Val Thr Ala Val 225 230 235 240

AGC GAC GGC ATG AAC TTC GTC TC 743 Ser Asp Gly Met Asn Phe Val

245 (2) INFORMATION FOR SEQ ID NO: 11:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 247 ammo acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Asp Val Met Trp Met His Asp Tyr His Leu Met Val Leu Pro Thr Phe 1 5 10 15 Leu Arg Arg Arg Phe Asn Arg Leu Arg Met Gly Phe Phe Leu His Ser 20 25 30

Pro Phe Pro Ser Ser Glu He Tyr Arg Thr Leu Pro Val Arg Glu Glu 35 40 45

He Leu Lys Ala Leu Leu Cys Ala Asp He Val Gly Phe His Thr Phe 50 55 60

Asp Tyr Ala Arg His Phe Leu Ser Cys Cys Ser Arg Met Leu Gly Leu 65 70 75 80

Glu Tyr Gin Ser Lys Arg Gly Tyr He Gly Leu Glu Tyr Tyr Gly Arg 85 90 95 Thr Val Gly He Lys He Met Pro Val Gly He His Met Gly His He 100 105 110

Glu Ser Met Lys Lys Leu Ala Ala Lys Glu Leu Met Leu Lys Ala Leu 115 120 125

Lys Gin Gin Phe Glu Gly Lys Thr Val Leu Leu Gly Ala Asp Asp Leu 130 135 140

Asp He Phe Lys Gly He Asn Leu Lys Leu Leu Ala Met Glu Gin Met 145 150 155 160

Leu Lys Gin His Pro Lys Trp Gin Gly Gin Ala Val Leu Val Gin He 165 170 175 Ala Asn Pro Thr Arg Gly Lys Gly Val Asp Phe Glu Glu He Gin Ala 180 185 190

Glu He Ser Glu Ser Cys Lys Arg He Asn Lys Gin Phe Gly Lys Pro 195 200 205

Gly Tyr Glu Pro He Val Tyr He Asp Arg Pro Val Ser Ser Ser Glu 210 215 220

Arg Met Ala Tyr Tyr Ser He Ala Glu Cys Val Val Val Thr Ala Val 225 230 235 240 y

Ser Asp Gly Met Asn Phe Val 245

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 395 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum (B) STRAIN: Samsun NN

(F) TISSUE TYPE: Leaf

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1..395

(D) OTHER INFORMATION: /partial

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GCG AAA CCG GTG ATG AAA CTT TAC AGG GAA GCA ACT GAC GGA TCA TAT 48

Ala Lys Pro Val Met Lys Leu Tyr Arg Glu Ala Thr Asp Gly Ser Tyr

1 5 10 15

ATA GAA ACT AAA GAG AGT GCA TTA GTG TGG CAC CAT CAT GAT GCA GAC 96 He Glu Thr Lys Glu Ser Ala Leu Val Trp His His His Asp Ala Asp 20 25 30

CCT GAC TTT GGC TCC TGC CAG GCA AAG GAA TTG TTG GAT CAT TTG GAA 144 Pro Asp Phe Gly Ser Cys Gin Ala Lys Glu Leu Leu Asp His Leu Glu 35 40 45

AGC GTA CTT GCA AAT GAA CCT GCA GTT GTT AAG AGG GGC CAA CAT ATT 192

Ser Val Leu Ala Asn Glu Pro Ala Val Val Lys Arg Gly Gin His He

50 55 60

GTT GAA GTC AAG CCA CAA GGT GTG ACC AAA GGA TTA GTT TCA GAG AAG 240

Val Glu Val Lys Pro Gin Gly Val Thr Lys Gly Leu Val Ser Glu Lys

65 70 75 80 GTT CTC TCG ATG ATG GTT GAT AGT GGG AAA CCG CCC GAT TTT GTT ATG 288 Val Leu Ser Met Met Val Asp Ser Gly Lys Pro Pro Asp Phe Val Met 85 90 95

TGC ATT GGA GAT GAT AGG TCA GAC GAA GAC ATG TTT GAG AGC ATA TTA 336 Cys He Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Ser He Leu 100 105 110 y

AGC ACC GTA TCC AGT CTG TCA GTC ACT GCT GCC CCT GAT GTC TTT GCC 384 Ser Thr Val Ser Ser Leu Ser Val Thr Ala Ala Pro Asp Val Phe Ala 115 120 125 TGC ACC GTC GG 395

Cys Thr Val 130

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 131 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Ala Lys Pro Val Met Lys Leu Tyr Arg Glu Ala Thr Asp Gly Ser Tyr 1 5 10 15

He Glu Thr Lys Glu Ser Ala Leu Val Trp His His His Asp Ala Asp 20 25 30

Pro Asp Phe Gly Ser Cys Gin Ala Lys Glu Leu Leu Asp His Leu Glu 35 40 45 Ser Val Leu Ala Asn Glu Pro Ala Val Val Lys Arg Gly Gin His He 50 55 60

Val Glu Val Lys Pro Gin Gly Val Thr Lys Gly Leu Val Ser Glu Lyε 65 70 75 80

Val Leu Ser Met Met Val Asp Ser Gly Lys Pro Pro Asp Phe Val Met 85 90 95

Cys He Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Ser He Leu 100 105 110

Ser Thr Val Ser Ser Leu Ser Val Thr Ala Ala Pro Asp Val Phe Ala 115 120 125 Cys Thr Val 130

( 2 ) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 491 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

[ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..491

(D) OTHER INFORMATION: /partial (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GGG CTG TCG GCG GAA CAC GGC TAT TTC TTG AGG ACG AGT CAA GAT GAA 48

Gly Leu Ser Ala Glu His Gly Tyr Phe Leu Arg Thr Ser Gin Asp Glu 1 5 10 15

GAA TGG GAA ACA TGT GTA CCA CCA GTG GAA TGT TGT TGG AAA GAA ATA 96

Glu Trp Glu Thr Cys Val Pro Pro Val Glu Cys Cys Trp Lys Glu He 20 25 30 GCT GAG CCT GTT ATG CAA CTT TAC ACT GAG ACT ACT GAT GGA TCA GTT 144

Ala Glu Pro Val Met Gin Leu Tyr Thr Glu Thr Thr Asp Gly Ser Val 35 40 45

ATT GAA GAT AAG GAA ACA TCA ATG GTC TGG TCT TAC GAG GAT GCG GAT 192 He Glu Asp Lys Glu Thr Ser Met Val Trp Ser Tyr Glu Asp Ala Asp

50 55 60

CCT GAT TTT GGA TCA TGT CAG GCT AAG GAA CTT CTT GAT CAC CTA GAA 240 Pro Asp Phe Gly Ser Cys Gin Ala Lys Glu Leu Leu Asp His Leu Glu 65 70 75 80

AGT GTA CTA GCT AAT GAA CCG GTC ACT GTC AGG AGT GGA CAG AAT ATA 288 Ser Val Leu Ala Aεn Glu Pro Val Thr Val Arg Ser Gly Gin Aεn He 85 90 95

GTG GAA GTT AAG CCC CAG GGT GTA TCC AAA GGG CTT GTT GCC AAG CGC 336 Val Glu Val Lys Pro Gin Gly Val Ser Lys Gly Leu Val Ala Lys Arg 100 105 110 CTG CTT TCC GCA ATG CAA GAG AAA GGA ATG TCA CCA GAT TTT GTC CTT 384 Leu Leu Ser Ala Met Gin Glu Lys Gly Met Ser Pro Asp Phe Val Leu 115 120 125

TGC ATA GGA GAT GAC CGA TCG GAT GAA GAC ATG TTC GAG GTG ATC ATG 432 Cys He Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Val He Met 130 135 140

AGC TCG ATG TCT GGC CCG TCC ATG GCT CCA ACA GCT GAA GTC TTT GCC 480 Ser Ser Met Ser Gly Pro Ser Met Ala Pro Thr Ala Glu Val Phe Ala 145 150 155 160 TGC ACC GTC GG 491

Cys Thr Val

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 163 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Gly Leu Ser Ala Glu His Gly Tyr Phe Leu Arg Thr Ser Gin Asp Glu 1 5 10 15

Glu Trp Glu Thr Cys Val Pro Pro Val Glu Cys Cys Trp Lys Glu He 20 25 30

Ala Glu Pro Val Met Gin Leu Tyr Thr Glu Thr Thr Asp Gly Ser Val 35 40 45 He Glu Asp Lys Glu Thr Ser Met Val Trp Ser Tyr Glu Asp Ala Asp 50 55 60

Pro Asp Phe Gly Ser Cys Gin Ala Lys Glu Leu Leu Asp His Leu Glu 65 70 75 80

Ser Val Leu Ala Asn Glu Pro Val Thr Val Arg Ser Gly Gin Asn He 85 90 95

Val Glu Val Lys Pro Gin Gly Val Ser Lys Gly Leu Val Ala Lys Arg 100 105 HO

Leu Leu Ser Ala Met Gin Glu Lys Gly Met Ser Pro Asp Phe Val Leu 115 120 125 Cys He Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Glu Val He Met 130 135 140

Ser Ser Met Ser Gly Pro Ser Met Ala Pro Thr Ala Glu Val Phe Ala 145 150 155 160

Cys Thr Val

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 361 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

TTTGATTATG ATGGGACGCT GCTGTCGGAG GAGAGTGTGG ACAAAACCCC GAGTGAAGAT 60

GACATCTCAA TTCTGAATGG TTTATGCAGT GATCCAAAGA ACGTAGTCTT TATCGTGAGT 120

GGCAGAGGAA AGGATACACT TAGCAAGTGG TTCTCTCCGT GTCCGAGACT CGGCCTATCA 180 GCAGAACATG GATATTTCAC TAGGTGGAGT AAGGATTCCG AGTGGGAATC TCGTCCATAG 240

CTGCAGACCT TGACTGGAAA AAAATAGTGT TGCCTATTAT GGAGCGCTAC ACAGAGCACA 300

GATGGTTCGT CGATAGAACA GAAGGAAACC TCGTGTTGGC TCATCAAATG CTGGCCCCGA 360

A 361

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GGAAACCCAC AGGATGTAAG CAAAGTTTTA GTTTTTGAGA TCTCTTGGCA TCAAOCAAAG 60

TAGAGGGAAG TCACCCGATT CGTGCTGTGC GTAGGGATGA CAGATCGGAC GACTTAGA 118 (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 417 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

TTGTGGCCGA TGTTCCACTA CATGTTGCCG TTCTCACCTG ACCATGGAGG CCGCTTTGAT 60

CGCTCTATGT GGGAAGCATA TGTTTCTGCC AACAAGTTGT TTTCACAAAA AGTAGTTGAG 120

GTTCTTAATC CTGAGGATGA CTTTGTCTGG ATTCATGATT ATCATTTGAT GGTGTTGCCA 180

ACGTTCTTGA GGAGGCGGTT CAATCGTTTG AGAATGGGGT TTTTCCTTCA CAGTCCATTC 240 CTTCATCTGA GATTTACAGG ACACTTCCTG TTAGAGAGGA AATACTCAAG GCTTTGCTCT 300

GTGCTGACAT TGTTGGATTC CACACTTTTG ACTACGCGAG ACACTTCCTC TCTTGTTGCA 360

GTCGATTTTG GGTAGAGTAC AGTCTAAAAA AAGTTATATT GGGTTAAAAT ACTATGG 417

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 411 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GGGTCATATT GATCCATGAA GAAATTGCAG CGAAAGAGTG ATGCTTTAAT GCGTAAAGCA 60 GCAATTTGAA GGGAAAACTG TGTTGTTAGG TGCCGATGAC CTGGATATTT TCAAAGGTAT 120

GAACTTAAAG CTTCTAGCTA TGGAACAGAT GCTCAAACAT CACCCCAAGT GGCAAGGGCA 180

GGCTGTGTTG GTCCAAGATT GCAAATCCTA CGAGGGGTAA AGGAGTAGAT TTTGACGAAA 240

TACGGCTGAG ACATCGGAAA GCTGTAAGAG AATCAATAAG CAATTCGGCA AGCCTGGATA 300

TGAGCCTATA GTTTATATTG ATAGGCCCGT GTCAAGCAGT GAACGCATGG CATATTACAG 360 TATTGCAGGA TGTGTTGTGG TCACGCTGTG AGCGATGGCA TGAATCTGTT C 411

(2) INFORMATION FOR SEQ ID NO: 20:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 405 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (n) MOLECULE TYPE: cDNA to mRNA (m) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf

(xi) SEQUENCE DESCRIPTION:^' SEQ ID NO: 20:

TGGGGTGGTT CCTGCATACG CCGTTTCCTT CTTCTGAGAT ATATAAAACT TTGCCTATTC 60 GCGAAAGATC TTACAGCTCT CTTGAATTCA ATTTGATTGG GTTCCACACT TTTGACTATG 120

CAGGCACTTC CTCTCGTGTT GCAGTCGGAT GTTAGGTATT TCTTATGATC AAAAAGGGGT 180

TACATAGGCC TCGATATTAT GGCAGGACTG TAATATAAAA ATTCTGCCAG CGGGTATTCA 240

TATGGGGCAG CTTCAGCAAG TCTTGAGTCT TCCTGAAACG GAGGCAAAAT CTCGGAACTC 300

GTGCAGCATT TAATCATCAG GGGGAGGACA TTGTTGCTGG GATTGATGAC TGGACATATT 360 TAAAGGCTCA TTTGAATTTA TTACCATGGA ACAACTCTAT TGCAC 405 ( 2 ) INFORMATION FOR SEQ ID NO : 21 :

( i ) SEQUENCE CHARACTERI STICS :

( A ) LENGTH : 427 base pairs ( B ) TYPE : nucl eic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

ATCATATGGG GCAGCTTCAG CAATCTTGAT CTTCCTGAAA CGGAGGCAAA AGTCTTCGGA 60

ACTCGGCAGC AGTTTAATCA TCAGGGGAGG ACATTGTTGC TGGGAGTTGA TGACATGGAC 120

ATATTTAAAG GCATCAGTTT GAAGTTATTA GCAATGGAAC AACTTCTATT GCAGCACCCG 180

GAGAAGCAGG GGAAGGTTGT TTTGGTGCAG ATAGCCAATC CTGCTAGAGG CAAAGGAAAA 240 GATGTCAAAG AAGTGCAGGA AGAAACTCAT TGACGGTGAA GCGAATTAAT GAAGCATTTG 300

GAAGACCTGG GTACGAACCA GTTATCTTGA TTGATAAGCC ACTAAAGTTT TATGAAAGGA 360

TTGCTTATTA TGTTGTTGCA GAGTGTTGCC TAGTCACTGC TGTCAGCGAT GGCATGAACC 420

TCGTCTC 427

(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 315 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf y>

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

GATGTGGATG CATGACTACC AATCCAAGAG GGGGTATATT GGTCTTGACT ATTATGGTAA 60 ACTGTGACCA TTAAAATCCT TCCAGTTGGT ATTCACATGG GACAACTCCA AAATGTTATG 120

TCACTACAGA CACGGGAAAG AAAGCAAAGG AGTTGAAAGA AAAATATGAG GGGAAAATTG 180

TGATGTTAGG TATTGATGAT ATGGACATGT TTAAAGGAAT TGGTCTAAAG TTTCTGGCAA 240

TGGGGAGGCT TCTAGATGAA AACCCTGTCT TGAGGGGTAA AGTGGTATTG GTTCAATCAC 300

CAGGCCTGGA AATTA 315 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 352 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA to mRNA (in) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Nicotiana tabacum

(B) STRAIN: Samsun NN (F) TISSUE TYPE: Leaf

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

AGAAGTAAAG GGAGTGAGTC CCCGAGGTTC AAAAAGAGGT CAACAGAATT GCAGTGAAAT 60

TAATAAAAAA TATGGCAAAC CGGGGTACAA GCCGATTGTT TGTATCAATG GTCCAGTTTC 120 GACACAAGAC AAGATTGCAC ATTATGCGGT CTTGAGTGTG TTGTTGTTAA TGCTGTTAGA 180

GATGGGATGA ACTTGGTGCC TTATGAGTAT ACGGTCTTTA GGCAGGGCAG CGATAATTTG 240

GATAAGGCCT TGCAGCTAGA TGGTCCTACT GCTTCCAGAA AGAGTGTGAT TATTGTCTTG 300

AATTCGTTGG GTGCTCGCCA TCTTTAGTGG CGCCATCCGC GTCAACCCCT GG 352

(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2640 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA ( m ) HYPOTHETICAL : NO ( m ) ANTI - SENSE : NO ( vi ) ORIGINAL SOURCE :

( A ) ORGANISM : Hel ianthus annuus ( F ) TISSUE TYPE : Lea f

( ix ) FEATURE : (A ) NAME/ KEY : CDS

(B) LOCATION: 171..2508

(ix) FEATURE:

(A) NAME/KEY: unsure (B) LOCATION: replace(2141..2151, "ccatnnntta" )

(ix) FEATURE:

(A) NAME/KEY: unsure

(B) LOCATION: replace(2237..2243, "actnaaa")

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

GGATCCTGCG GTTTCATCAC ACAATATGAT ACTGTTACAT CTGATGCCCC TTCAGATGTC 60 CCAAATAGGT TGATTGTCGT ATCGAATCAG TTACCCATAA TCGCTAGGCT AAGACTAACG 120

ACAATGGAGG GTCCTTTTGG GATTTCACTT GGGACGAGAG TTCGATTTAC ATG CAC 176

Met His 1

ATC AAA GAT GCA TTA CCC GCA GCC GTT GAG GTT TTC TAT GTT GGC GCA 224 He Lys Asp Ala Leu Pro Ala Ala Val Glu Val Phe Tyr Val Gly Ala 5 10 15 CTA AGG GCT GAC GTT GGC CCT ACC GAA CAA GAT GAC GTG TCA AAG ACA 272

Leu Arg Ala Asp Val Gly Pro Thr Glu Gin Asp Asp Val Ser Lys Thr 20 25 30

TTG CTC GAT AGG TTT AAT TGC GTT GCG GTT TTT GTC CCT ACT TCA AAA 320 Leu Leu Asp Arg Phe Asn Cys Val Ala Val Phe Val Pro Thr Ser Lys 35 40 45 50

TGG GAC CAA TAT TAT CAC TGC TTT TGT AAG CAG TAT TTG TGG CCG ATA 368 Trp Asp Gin Tyr Tyr His Cys Phe Cys Lys Gin Tyr Leu Trp Pro He 55 60 65

TTT CAT TAC AAG GTT CCC GCT TCT GAC GTC AAG AGT GTC CCG AAT AGT 416 Phe His Tyr Lys Val Pro Ala Ser Asp Val Lys Ser Val Pro Asn Ser 70 75 80

CGG GAT TCA TGG AAC GCT TAT GTT CAC GTG AAC AAA GAG TTT TCC CAG 464 Arg Asp Ser Trp Asn Ala Tyr Val His Val Asn Lys Glu Phe Ser Gin 85 90 95 /oo

AAG GTG ATG GAG GCA GTA ACC AAT GCT AGC AAT TAT GTA TGG ATA CAT 512

Lys Val Met Glu Ala Val Thr Asn Ala Ser Asn Tyr Val Trp He His

100 105 110 GAC TAC CAT TTA ATG ACG CTA CCG ACT TTC TTG AGG CGG GAT TTT TGT 560

Asp Tyr His Leu Met Thr Leu Pro Thr Phe Leu Arg Arg Asp Phe Cys

115 120 125 130

CGT TTT AAA ATC GGT TTT TTT CTG CAT AGC CCG TTT CCT TCC TCG GAG 608 Arg Phe Lys He Gly Phe Phe Leu His Ser Pro Phe Pro Ser Ser Glu

135 140 145

GTT TAC AAG ACC CTA CCA ATG AGA AAC GAG CTC TTG AAG GGT CTG TTA 656

Val Tyr Lys Thr Leu Pro Met Arg Asn Glu Leu Leu Lys Gly Leu Leu 150 155 160

AAT GCT GAT CTT ATC GGG TTC CAT ACA TAC GAT TAT GCC CGT CAT TTT 704

Asn Ala Asp Leu He Gly Phe His Thr Tyr Asp Tyr Ala Arg His Phe

165 170 175

CTA ACG TGT TGT AGT CGA ATG TTT GGT TTG GAT CAT CAG TTG AAA AGG 752

Leu Thr Cys Cys Ser Arg Met Phe Gly Leu Asp His Gin Leu Lys Arg

180 185 190 GGG TAC ATT TTC TTG GAA TAT AAT GGA AGG AGC ATT GAG ATC AAG ATA 800

Gly Tyr He Phe Leu Glu Tyr Asn Gly Arg Ser He Glu He Lys He

195 200 205 210

AAG GCG AGC GGG ATT CAT GTT GGT CGA ATG GAG TCG TAC TTG AGT CAG 848 Lys Ala Ser Gly He His Val Gly Arg Met Glu Ser Tyr Leu Ser Gin

215 220 225

CCC GAT ACA AGA TTA CAA GTT CAA GAA CTA AAA AAA CGT TTC GAA GGG 896

Pro Asp Thr Arg Leu Gin Val Gin Glu Leu Lys Lys Arg Phe Glu Gly 230 235 240

AAA ATC GTG CTA CTT GGA GTT GAT GAT TTG GAT ATA TTC AAA GGT GTG 944

Lys He Val Leu Leu Gly Val Asp Asp Leu Asp He Phe Lys Gly Val

245 250 255

AAC TTC AAG GTT TTA GCG TTG GAG AAG TTA CTT AAA TCA CAC CCG AGT 992

Asn Phe Lys Val Leu Ala Leu Glu Lys Leu Leu Lys Ser His Pro Ser

260 265 270 TGG CAA GGG CGT GTG GTT TTG GTG CAA ATC TTG AAT CCC GCT CGC GCG 1040

Trp Gin Gly Arg Val Val Leu Val Gin He Leu Asn Pro Ala Arg Ala

275 280 285 290

CGT TGC CAA GAC GTC GAT GAG ATC AAT GCC GAG ATA AGA ACA GTC TGT 1088 Arg Cys Gin Asp Val Asp Glu He Asn Ala Glu He Arg Thr Val Cys

295 300 305

GAA AGA ATC AAT AAC GAA CTG GGA AGC CCG GGA TAC CAG CCC GTT GTG 1136

Glu Arg He Asn Asn Glu Leu Gly Ser Pro Gly Tyr Gin Pro Val Val 310 315 320 l o t

TTA ATT GAT GGG CCC GTT TCG TTA AGT GAA AAA GCT GCT TAT TAT GCT 1184 Leu He Asp Gly Pro Val Ser Leu Ser Glu Lys Ala Ala Tyr Tyr Ala 325 330 335 ATC GCC GAT ATG GCA ATT GTT ACA CCG TTA CGT GAC GGC ATG AAT CTT 1232 He Ala Asp Met Ala He Val Thr Pro Leu Arg Asp Gly Met Asn Leu 340 345 350

ATC CCG TAC GAG TAC GTC GTT TCC CGA CAA AGT GTT AAT GAC CCA AAT 1280 He Pro Tyr Glu Tyr Val Val Ser Arg Gin Ser Val Asn Asp Pro Asn 355 360 365 370

CCC AAT ACT CCA AAA AAG AGC ATG CTA GTG GTC TCC GAG TTC ATC GGG 1328 Pro Asn Thr Pro Lys Lys Ser Met Leu Val Val Ser Glu Phe He Gly 375 380 385

TGT TCA CTA TCT TTA ACC GGG GCC ATA CGG GTC AAC CCA TGG GAT GAG 1376

Cys Ser Leu Ser Leu Thr Gly Ala He Arg Val Asn Pro Trp Asp Glu

390 395 400

TTG GAG ACA GCA GAA GCA TTA TAC GAC GCA CTC ATG GCT CCT GAT GAC 1424

Leu Glu Thr Ala Glu Ala Leu Tyr Asp Ala Leu Met Ala Pro Asp Asp

405 410 415 CAT AAA GAA ACC GCC CAC ATG AAA CAG TAT CAA TAC ATT ATC TCC CAT 1472

His Lys Glu Thr Ala His Met Lys Gin Tyr Gin Tyr He He Ser His 420 425 430

GAT GTA GCT AAC TGG GCT CGT AGC TTC TTT CAA GAT TTA GAG CAA GCG 1520 Asp Val Ala Asn Trp Ala Arg Ser Phe Phe Gin Asp Leu Glu Gin Ala

435 440 445 450

TGC ATC GAT CAT TCT CGT AAA CGA TGC ATG AAT TTA GGA TTT GGG TTA 1568 Cys He Asp His Ser Arg Lys Arg Cys Met Asn Leu Gly Phe Gly Leu 455 460 465

GAT ACT AGA GTC GTT CTT TTT GAT GAG AAG TTT AGC AAG TTG GAT ATA 1616 Asp Thr Arg Val Val Leu Phe Asp Glu Lys Phe Ser Lys Leu Asp He 470 475 480

GAT GTC TTG GAG AAT GCT TAT TCC ATG GCT CAA AAT CGG GCC ATA CTT 1664 Asp Val Leu Glu Asn Ala Tyr Ser Met Ala Gin Asn Arg Ala He Leu 485 490 495 TTG GAC TAT GAC GGC ACT GTT ACT CCA TCT ATC AGT AAA TCT CCA ACT 1712

Leu Asp Tyr Asp Gly Thr Val Thr Pro Ser He Ser Lys Ser Pro Thr 500 505 510

GAA GCT GTT ATC TCC ATG ATC AAC AAA CTG TGC AAT GAT CCA AAG AAC 1760 Glu Ala Val He Ser Met He Asn Lys Leu Cys Asn Asp Pro Lys Asn 515 520 525 530

ATG GTG TTC ATC GTT AGT GGA CGC AGT AGA GAA AAT CTT GGC AGT TGG 1808 Met Val Phe He Val Ser Gly Arg Ser Arg Glu Asn Leu Gly Ser Trp 535 540 545 TTC GGC GCG TGT GAG AAA CCC GCC ATT GCA GCT GAG CAC GGA TAC TTT 1856 Phe Gly Ala Cys Glu Lys Pro Ala He Ala Ala Glu His Gly Tyr Phe 550 555 560 ATA AGG TGG GCG GGT GAT CAA GAA TGG GAA ACG TGC GCA CGT GAG AAT 1904 He Arg Trp Ala Gly Asp Gin Glu Trp Glu Thr Cys Ala Arg Glu Asn 565 570 575

AAT GTC GGG TGG ATG GAA ATG GCT GAG CCG GTT ATG AAT CTT TAT ACA 1952 Asn Val Gly Trp Met Glu Met Ala Glu Pro Val Met Asn Leu Tyr Thr 580 585 590

GAA ACT ACT GAC GGT TCG TAT ATT GAA AAG AAA GAA ACT GCA ATG GTT 2000 Glu Thr Thr Asp Gly Ser Tyr He Glu Lys Lys Glu Thr Ala Met Val 595 600 605 610

TGG CAC TAT GAA GAT GCT GAT AAA GAT CTT GGG TTG GAG CAG GCT AAG 2048

Trp His Tyr Glu Asp Ala Asp Lys Asp Leu Gly Leu Glu Gin Ala Lys 615 620 625

GAA CTG TTG GAC CAT CTT GAA AAC GTG CTC GCT AAT GAG CCC GTT GAA 2096

Glu Leu Leu Asp His Leu Glu Asn Val Leu Ala Asn Glu Pro Val Glu 630 635 640 GTG AAA CGA GGT CAA TAC ATT GTA GAA GTT AAA CCA CAG GTA CCC CAT 2144

Val Lys Arg Gly Gin Tyr He Val Glu Val Lys Pro Gin Val Pro His 645 650 655

GGG TTA CCT TCT TGT TAT GAC ATT CAT AGG CAC AGA TTT GTA GAA TCT 2192 Gly Leu Pro Ser Cys Tyr Asp He His Arg His Arg Phe Val Glu Ser

660 665 670

TTT AAC TTA AAT TTC TTT AAA TAT GAA TGC AAT TAT AGG GGG TCA CTG 2240 Phe Asn Leu Asn Phe Phe Lys Tyr Glu Cys Asn Tyr Arg Gly Ser Leu 675 680 685 690

AAA GGT ATA GTT GCA GAG AAG ATT TTT GCG TTC ATG GCT GAA AAG GGA 2288

Lys Gly He Val Ala Glu Lys He Phe Ala Phe Met Ala Glu Lys Gly

695 700 705

AAA CAG GCT GAT TTC GTG TTG AGC GTT GGA GAT GAT AGA AGT GAT GAA 2336

Lys Gin Ala Asp Phe Val Leu Ser Val Gly Asp Asp Arg Ser Asp Glu

710 715 720 GAC ATG TTT GTG GCC ATT GGG GAT GGA ATA AAA AAG GGT CGG ATA ACT 2384 Asp Met Phe Val Ala He Gly Asp Gly He Lys Lys Gly Arg He Thr 725 730 735

AAC AAC AAT TCA GTG TTT ACA TGC GTA GTG GGA GAG AAA CCG AGT GCA 2432 Asn Asn Asn Ser Val Phe Thr Cys Val Val Gly Glu Lys Pro Ser Ala 740 745 750

GCT GAG TAC TTT TTA GAC GAG ACG AAA GAT GTT TCA ATG ATG CTC GAG 2480 Ala Glu Tyr Phe Leu Asp Glu Thr Lys Asp Val Ser Met Met Leu Glu 755 760 765 770 AAG CTC GGG TGT CTC AGC AAC CAA GGA T GATGATCCGG AAGCTTCTCG 2528 Lys Leu Gly Cys Leu Ser Asn Gin Gly 775 TGATCTTTAT GAGTTAAAAG TTTTCGACTT TTTCTTCATC AAGATTCATG GGAAAGTTGT 2588

TCAATATGAA CTTGTGTTTC TTGGTTCTGG ATTTTAGGGA GTCTATGGAT CC 2640

(2) INFORMATION FOR SEQ ID NO. 25-

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH 779 ammo acids (D) TOPOLOGY, linear

(n) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO 25-

Met His He Lys Asp Ala Leu Pro Ala Ala Val Glu Val Phe Tyr Val 1 5 10 15

Gly Ala Leu Arg Ala Asp Val Gly Pro Thr Glu Gin Asp Asp Val Ser 20 25 30

Lys Thr Leu Leu Asp Arg Phe Asn Cys Val Ala Val Phe Val Pro Thr 35 40 45 Ser Lys Trp Asp Gin Tyr Tyr His Cys Phe Cys Lys Gin Tyr Leu Trp 50 55 60

Pro He Phe His Tyr Lys Val Pro Ala Ser Asp Val Lys Ser Val Pro 65 70 75 80

Asn Ser Arg Asp Ser Trp Asn Ala Tyr Val His Val Asn Lys Glu Phe 85 90 95

Ser Gin Lys Val Met Glu Ala Val Thr Asn Ala Ser Asn Tyr Val Trp 100 105 110

He His Asp Tyr His Leu Met Thr Leu Pro Thr Phe Leu Arg Arg Asp 115 120 125 Phe Cys Arg Phe Lys He Gly Phe Phe Leu His Ser Pro Phe Pro Ser 130 135 140

Ser Glu Val Tyr Lys Thr Leu Pro Met Arg Asn Glu Leu Leu Lys Gly 145 150 155 160

Leu Leu Asn Ala Asp Leu He Gly Phe His Thr Tyr Asp Tyr Ala Arg 165 170 175

His Phe Leu Thr Cys Cys Ser Arg Met Phe Gly Leu Asp His Gin Leu 180 185 190 Lyε Arg Gly Tyr He Phe Leu Glu Tyr Asn Gly Arg Ser He Glu He 195 200 205

Lys He Lys Ala Ser Gly He His Val Gly Arg Met Glu Ser Tyr Leu 210 215 220

Ser Gin Pro Asp Thr Arg Leu Gin Val Gin Glu Leu Lys Lys Arg Phe 225 230 235 240 Glu Gly Lys He Val Leu Leu Gly Val Asp Asp Leu Asp He Phe Lys

245 250 255

Gly Val Asn Phe Lys Val Leu Ala Leu Glu Lys Leu Leu Lys Ser His 260 265 270

Pro Ser Trp Gin Gly Arg Val Val Leu Val Gin He Leu Asn Pro Ala 275 280 285

Arg Ala Arg Cys Gin Asp Val Asp Glu He Asn Ala Glu He Arg Thr 290 295 300

Val Cys Glu Arg He Asn Asn Glu Leu Gly Ser Pro Gly Tyr Gin Pro 305 310 315 320 Val Val Leu He Asp Gly Pro Val Ser Leu Ser Glu Lys Ala Ala Tyr

325 330 335

Tyr Ala He Ala Asp Met Ala He Val Thr Pro Leu Arg Asp Gly Met 340 345 350

Asn Leu He Pro Tyr Glu Tyr Val Val Ser Arg Gin Ser Val Asn Asp 355 360 365

Pro Asn Pro Asn Thr Pro Lys Lys Ser Met Leu Val Val Ser Glu Phe 370 375 380

He Gly Cys Ser Leu Ser Leu Thr Gly Ala He Arg Val Asn Pro Trp 385 390 395 400 Asp Glu Leu Glu Thr Ala Glu Ala Leu Tyr Asp Ala Leu Met Ala Pro

405 410 415

Asp Asp His Lys Glu Thr Ala His Met Lys Gin Tyr Gin Tyr He He 420 425 430

Ser His Asp Val Ala Asn Trp Ala Arg Ser Phe Phe Gin Asp Leu Glu 435 440 445

Gin Ala Cys He Asp His Ser Arg Lys Arg Cys Met Asn Leu Gly Phe 450 455 460

Gly Leu Asp Thr Arg Val Val Leu Phe Asp Glu Lys Phe Ser Lys Leu 465 470 475 480 Asp He Asp Val Leu Glu Asn Ala Tyr Ser Met Ala Gin Asn Arg Ala

485 490 495 ' 05

He Leu Leu Asp Tyr Asp Gly Thr Val Thr Pro Ser He Ser Lys Ser 500 505 510

Pro Thr Glu Ala Val He Ser Met He Asn Lys Leu Cys Asn Asp Pro 515 520 525

Lys Asn Met Val Phe He Val Ser Gly Arg Ser Arg Glu Asn Leu Gly 530 535 540 Ser Trp Phe Gly Ala Cys Glu Lys Pro Ala He Ala Ala Glu His Gly 545 550 555 560

Tyr Phe He Arg Trp Ala Gly Asp Gin Glu Trp Glu Thr Cys Ala Arg 565 570 575

Glu Asn Asn Val Gly Trp Met Glu Met Ala Glu Pro Val Met Asn Leu 580 585 590

Tyr Thr Glu Thr Thr Asp Gly Ser Tyr He Glu Lys Lys Glu Thr Ala 595 600 605

Met Val Trp His Tyr Glu Asp Ala Asp Lys Asp Leu Gly Leu Glu Gin 610 615 620 Ala Lys Glu Leu Leu Asp His Leu Glu Asn Val Leu Ala Asn Glu Pro 625 630 635 640

Val Glu Val Lys Arg Gly Gin Tyr He Val Glu Val Lys Pro Gin Val 645 650 655

Pro His Gly Leu Pro Ser Cys Tyr Asp He His Arg His Arg Phe Val 660 665 670

Glu Ser Phe Asn Leu Asn Phe Phe Lys Tyr Glu Cys Asn Tyr Arg Gly 675 680 685

Ser Leu Lys Gly He Val Ala Glu Lys He Phe Ala Phe Met Ala Glu 690 695 700 Lys Gly Lys Gin Ala Asp Phe Val Leu Ser Val Gly Asp Asp Arg Ser 705 710 715 720

Asp Glu Asp Met Phe Val Ala He Gly Asp Gly He Lys Lys Gly Arg 725 730 735

He Thr Asn Asn Asn Ser Val Phe Thr Cys Val Val Gly Glu Lys Pro 740 745 750

Ser Ala Ala Glu Tyr Phe Leu Asp Glu Thr Lys Asp Val Ser Met Met 755 760 765

Leu Glu Lys Leu Gly Cys Leu Ser Asn Gin Gly 770 775 (2) INFORMATION FOR SEQ ID NO: 26:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2130 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA to mRNA

(ill) HYPOTHETICAL: NO

(m) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Helianthus annuus

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 171..2130

(D) OTHER INFORMATION: /partial

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 26: GGATCCTGCG GTTTCATCAC ACAATATGAT ACTGTTACAT CTGATGCCCC TTCAGATGTC 60

CCAAATAGGT TGATTGTCGT ATCGAATCAG TTACCCATAA TCGCTAGGCT AAGACTAACG 120

ACAATGGAGG GTCCTTTTGG GATTTCACTT GGGACGAGAG TTCGATTTAC ATG CAC 176

1

ATC AAA GAT GCA TTA CCC GCA GCC GTT GAG GTT TTC TAT GTT GGC GCA 224 He Lys Asp Ala Leu Pro Ala Ala Val Glu Val Phe Tyr Val Gly Ala 5 10 15

CTA AGG GCT GAC GTT GGC CCT ACC GAA CAA GAT GAC GTG TCA AAG ACA 272

Leu Arg Ala Asp Val Gly Pro Thr Glu Gin Asp Asp Val Ser Lyε Thr

20 25 30

TTG CTC GAT AGG TTT AAT TGC GTT GCG GTT TTT GTC CCT ACT TCA AAA 320

Leu Leu Asp Arg Phe Asn Cys Val Ala Val Phe Val Pro Thr Ser Lys

35 40 45 50 TGG GAC CAA TAT TAT CAC TGC TTT TGT AAG CAG TAT TTG TGG CCG ATA 368 Trp Asp Gin Tyr Tyr His Cys Phe Cys Lys Gin Tyr Leu Trp Pro He 55 60 65

TTT CAT TAC AAG GTT CCC GCT TCT GAC GTC AAG AGT GTC CCG AAT AGT 416 Phe His Tyr Lys Val Pro Ala Ser Asp Val Lys Ser Val Pro Asn Ser 70 75 80

CGG GAT TCA TGG AAC GCT TAT GTT CAC GTG AAC AAA GAG TTT TCC CAG 464 Arg Asp Ser Trp Asn Ala Tyr Val His Val Asn Lys Glu Phe Ser Gin 85 90 95 / o y

AAG GTG ATG GAG GCA GTA ACC AAT GCT AGC AAT TAT GTA TGG ATA CAT 512

Lys Val Met Glu Ala Val Thr Asn Ala Ser Asn Tyr Val Trp He His

100 105 110 GAC TAC CAT TTA ATG ACG CTA CCG ACT TTC TTG AGG CGG GAT TTT TGT 560

Asp Tyr His Leu Met Thr Leu Pro Thr Phe Leu Arg Arg Asp Phe Cys 115 120 125 130

CGT TTT AAA ATC GGT TTT TTT CTG CAT AGC CCG TTT CCT TCC TCG GAG 608 Arg Phe Lys He Gly Phe Phe Leu His Ser Pro Phe Pro Ser Ser Glu

135 140 145

GTT TAC AAG ACC CTA CCA ATG AGA AAC GAG CTC TTG AAG GGT CTG TTA 656

Val Tyr Lys Thr Leu Pro Met Arg Asn Glu Leu Leu Lys Gly Leu Leu 150 155 160

AAT GCT GAT CTT ATC GGG TTC CAT ACA TAC GAT TAT GCC CGT CAT TTT 704 Asn Ala Asp Leu He Gly Phe His Thr Tyr Asp Tyr Ala Arg His Phe 165 170 175

CTA ACG TGT TGT AGT CGA ATG TTT GGT TTG GAT CAT CAG TTG AAA AGG 752

Leu Thr Cys Cys Ser Arg Met Phe Gly Leu Asp His Gin Leu Lys Arg

180 185 190 GGG TAC ATT TTC TTG GAA TAT AAT GGA AGG AGC ATT GAG ATC AAG ATA 800

Gly Tyr He Phe Leu Glu Tyr Asn Gly Arg Ser He Glu He Lys He 195 200 205 210

AAG GCG AGC GGG ATT CAT GTT GGT CGA ATG GAG TCG TAC TTG AGT CAG 848 Lys Ala Ser Gly He His Val Gly Arg Met Glu Ser Tyr Leu Ser Gin

215 220 225

CCC GAT ACA AGA TTA CAA GTT CAA GAA CTA AAA AAA CGT TTC GAA GGG 896

Pro Asp Thr Arg Leu Gin Val Gin Glu Leu Lys Lys Arg Phe Glu Gly 230 235 240

AAA ATC GTG CTA CTT GGA GTT GAT GAT TTG GAT ATA TTC AAA GGT GTG 944

Lys He Val Leu Leu Gly Val Asp Asp Leu Asp He Phe Lys Gly Val 245 250 255

AAC TTC AAG GTT TTA GCG TTG GAG AAG TTA CTT AAA TCA CAC CCG AGT 992

Asn Phe Lys Val Leu Ala Leu Glu Lys Leu Leu Lys Ser His Pro Ser

260 265 270 TGG CAA GGG CGT GTG GTT TTG GTG CAA ATC TTG AAT CCC GCT CGC GCG 1040

Trp Gin Gly Arg Val Val Leu Val Gin He Leu Asn Pro Ala Arg Ala 275 280 285 290

CGT TGC CAA GAC GTC GAT GAG ATC AAT GCC GAG ATA AGA ACA GTC TGT 1088 Arg Cys Gin Asp Val Asp Glu He Asn Ala Glu He Arg Thr Val Cys

295 300 305

GAA AGA ATC AAT AAC GAA CTG GGA AGC CCG GGA TAC CAG CCC GTT GTG 1136

Glu Arg He Asn Asn Glu Leu Gly Ser Pro Gly Tyr Gin Pro Val Val 310 315 320 t o£

TTA ATT GAT GGG CCC GTT TCG TTA AGT GAA AAA GCT GCT TAT TAT GCT 1184 Leu He Asp Gly Pro Val Ser Leu Ser Glu Lys Ala Ala Tyr Tyr Ala 325 330 335 ATC GCC GAT ATG GCA ATT GTT ACA CCG TTA CGT GAC GGC ATG AAT CTT 1232 He Ala Asp Met Ala He Val Thr Pro Leu Arg Asp Gly Met Asn Leu 340 345 350

ATC CCG TAC GAG TAC GTC GTT TCC CGA CAA AGT GTT AAT GAC CCA AAT 1280 He Pro Tyr Glu Tyr Val Val Ser Arg Gin Ser Val Aεn Asp Pro Asn 355 360 365 370

CCC AAT ACT CCA AAA AAG AGC ATG CTA GTG GTC TCC GAG TTC ATC GGG 1328 Pro Asn Thr Pro Lys Lys Ser Met Leu Val Val Ser Glu Phe He Gly 375 380 385

TGT TCA CTA TCT TTA ACC GGG GCC ATA CGG GTC AAC CCA TGG GAT GAG 1376

Cys Ser Leu Ser Leu Thr Gly Ala He Arg Val Asn Pro Trp Asp Glu 390 395 400

TTG GAG ACA GCA GAA GCA TTA TAC GAC GCA CTC ATG GCT CCT GAT GAC 1424

Leu Glu Thr Ala Glu Ala Leu Tyr Asp Ala Leu Met Ala Pro Aεp Asp 405 410 415 CAT AAA GAA ACC GCC CAC ATG AAA CAG TAT CAA TAC ATT ATC TCC CAT 1472 His Lys Glu Thr Ala His Met Lys Gin Tyr Gin Tyr He He Ser His 420 425 430

GAT GTA GCT AAC TGG GCT CGT AGC TTC TTT CAA GAT TTA GAG CAA GCG 1520 Asp Val Ala Asn Trp Ala Arg Ser Phe Phe Gin Asp Leu Glu Gin Ala 435 440 445 450

TGC ATC GAT CAT TCT CGT AAA CGA TGC ATG AAT TTA GGA TTT GGG TTA 1568 Cys He Asp His Ser Arg Lys Arg Cys Met Asn Leu Gly Phe Gly Leu 455 460 465

GAT ACT AGA GTC GTT CTT TTT GAT GAG AAG TTT AGC AAG TTG GAT ATA 1616

Asp Thr Arg Val Val Leu Phe Asp Glu Lys Phe Ser Lys Leu Asp He

470 475 480

GAT GTC TTG GAG AAT GCT TAT TCC ATG GCT CAA AAT CGG GCC ATA CTT 1664

Aεp Val Leu Glu Asn Ala Tyr Ser Met Ala Gin Asn Arg Ala He Leu 485 490 495 TTG GAC TAT GAC GGC ACT GTT ACT CCA TCT ATC AGT AAA TCT CCA ACT 1712 Leu Asp Tyr Asp Gly Thr Val Thr Pro Ser He Ser Lys Ser Pro Thr 500 505 510

GAA GCT GTT ATC TCC ATG ATC AAC AAA CTG TGC AAT GAT CCA AAG AAC 1760 Glu Ala Val He Ser Met He Asn Lys Leu Cys Asn Asp Pro Lys Asn 515 520 525 530

ATG GTG TTC ATC GTT AGT GGA CGC AGT AGA GAA AAT CTT GGC AGT TGG 1808 Met Val Phe He Val Ser Gly Arg Ser Arg Glu Asn Leu Gly Ser Trp 535 540 545 ⁵!)

TTC GGC GCG TGT GAG AAA CCC GCC ATT GCA GCT GAG CAC GGA TAC TTT 1856

Phe Gly Ala Cys Glu Lys Pro Ala He Ala Ala Glu His Gly Tyr Phe

550 555 560 ATA AGG TGG GCG GGT GAT CAA GAA TGG GAA ACG TGC GCA CGT GAG AAT 1904

He Arg Trp Ala Gly Asp Gin Glu Trp Glu Thr Cys Ala Arg Glu Asn

565 570 575

AAT GTC GGG TGG ATG GAA ATG GCT GAG CCG GTT ATG AAT CTT TAT ACA 1952 Asn Val Gly Trp Met Glu Met Ala Glu Pro Val Met Asn Leu Tyr Thr

580 585 590

GAA ACT ACT GAC GGT TCG TAT ATT GAA AAG AAA GAA ACT GCA ATG GTT 2000

Glu Thr Thr Asp Gly Ser Tyr He Glu Lys Lys Glu Thr Ala Met Val 595 600 605 610

TGG CAC TAT GAA GAT GCT GAT AAA GAT CTT GGG TTG GAG CAG GCT AAG 2048

Trp His Tyr Glu Asp Ala Asp Lys Asp Leu Gly Leu Glu Gin Ala Lys 615 620 625

GAA CTG TTG GAC CAT CTT GAA AAC GTG CTC GCT AAT GAG CCC GTT GAA 2096

Glu Leu Leu Asp His Leu Glu Asn Val Leu Ala Asn Glu Pro Val Glu

630 635 640 GTG AAA CGA GGT CAA TAC ATT GTA GAA GTT AAA C 2130

Val Lys Arg Gly Gin Tyr He Val Glu Val Lys

645 650

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 653 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Met His He Lys Asp Ala Leu Pro Ala Ala Val Glu Val Phe Tyr Val 1 5 10 15

Gly Ala Leu Arg Ala Asp Val Gly Pro Thr Glu Gin Asp Asp Val Ser 20 25 30

Lys Thr Leu Leu Asp Arg Phe Asn Cys Val Ala Val Phe Val Pro Thr 35 40 45

Ser Lys Trp Asp Gin Tyr Tyr His Cys Phe Cys Lys Gin Tyr Leu Trp 50 55 60

Pro He Phe His Tyr Lys Val Pro Ala Ser Asp Val Lys Ser Val Pro

65 70 75 80 Asn Ser Arg Asp Ser Trp Asn Ala Tyr Val His Val Asn Lys Glu Phe

85 90 95 4

/ /O

Ser Gin Lys Val Met Glu Ala Val Thr Asn Ala Ser Asn Tyr Val Trp 100 105 110

He His Asp Tyr His Leu Met Thr Leu Pro Thr Phe Leu Arg Arg Asp 115 120 125

Phe Cys Arg Phe Lys He Gly Phe Phe Leu His Ser Pro Phe Pro Ser 130 135 140 Ser Glu Val Tyr Lys Thr Leu Pro Met Arg Asn Glu Leu Leu Lys Gly 145 150 155 160

Leu Leu Asn Ala Aεp Leu He Gly Phe His Thr Tyr Asp Tyr Ala Arg 165 170 175

His Phe Leu Thr Cys Cys Ser Arg Met Phe Gly Leu Asp His Gin Leu 180 185 190

Lys Arg Gly Tyr He Phe Leu Glu Tyr Asn Gly Arg Ser He Glu He 195 200 205

Lys He Lys Ala Ser Gly He His Val Gly Arg Met Glu Ser Tyr Leu 210 215 220 Ser Gin Pro Asp Thr Arg Leu Gin Val Gin Glu Leu Lys Lys Arg Phe 225 230 235 240

Glu Gly Lys He Val Leu Leu Gly Val Asp Asp Leu Asp He Phe Lys 245 250 255

Gly Val Asn Phe Lys Val Leu Ala Leu Glu Lys Leu Leu Lys Ser His 260 265 270

Pro Ser Trp Gin Gly Arg Val Val Leu Val Gin He Leu Asn Pro Ala 275 280 285

Arg Ala Arg Cys Gin Asp Val Asp Glu He Asn Ala Glu He Arg Thr 290 295 300 Val Cys Glu Arg He Asn Asn Glu Leu Gly Ser Pro Gly Tyr Gin Pro 305 310 315 320

Val Val Leu He Asp Gly Pro Val Ser Leu Ser Glu Lys Ala Ala Tyr 325 330 335

Tyr Ala He Ala Asp Met Ala He Val Thr Pro Leu Arg Asp Gly Met 340 345 350

Asn Leu He Pro Tyr Glu Tyr Val Val Ser Arg Gin Ser Val Asn Asp 355 360 365

Pro Asn Pro Asn Thr Pro Lys Lys Ser Met Leu Val Val Ser Glu Phe 370 375 380 He Gly Cys Ser Leu Ser Leu Thr Gly Ala He Arg Val Asn Pro Trp 385 390 395 400 / / /

Asp Glu Leu Glu Thr Ala Glu Ala Leu Tyr Asp Ala Leu Met Ala Pro 405 410 415

Asp Asp His Lys Glu Thr Ala His Met Lys Gin Tyr Gin Tyr He He 420 425 430

Ser His Asp Val Ala Asn Trp Ala Arg Ser Phe Phe Gin Asp Leu Glu 435 440 445 Gin Ala Cys He Asp His Ser Arg Lys Arg Cys Met Asn Leu Gly Phe 450 455 460

Gly Leu Asp Thr Arg Val Val Leu Phe Asp Glu Lys Phe Ser Lys Leu 465 470 475 480

Asp He Asp Val Leu Glu Asn Ala Tyr Ser Met Ala Gin Asn Arg Ala 485 490 495

He Leu Leu Asp Tyr Asp Gly Thr Val Thr Pro Ser He Ser Lys Ser 500 505 510

Pro Thr Glu Ala Val He Ser Met He Asn Lys Leu Cys Asn Asp Pro 515 520 525 Lys Asn Met Val Phe He Val Ser Gly Arg Ser Arg Glu Asn Leu Gly 530 535 540

Ser Trp Phe Gly Ala Cys Glu Lys Pro Ala He Ala Ala Glu His Gly 545 550 555 560

Tyr Phe He Arg Trp Ala Gly Asp Gin Glu Trp Glu Thr Cys Ala Arg 565 570 575

Glu Asn Asn Val Gly Trp Met Glu Met Ala Glu Pro Val Met Asn Leu 580 585 590

Tyr Thr Glu Thr Thr Asp Gly Ser Tyr He Glu Lys Lys Glu Thr Ala 595 600 605 Met Val Trp His Tyr Glu Asp Ala Asp Lys Asp Leu Gly Leu Glu Gin

610 615 620

Ala Lys Glu Leu Leu Asp His Leu Glu Asn Val Leu Ala Asn Glu Pro 625 630 635 640

Val Glu Val Lys Arg Gly Gin Tyr He Val Glu Val Lys 645 650

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 390 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear /⁾ 2_

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Helianthuε annuus (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 3..258

(D) OTHER INFORMATION: /partial (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

TT GCA GAG AAG ATT TTT GCG TTC ATG GCT GAA AAG GGA AAA CAG GCT 47 Ala Glu Lys He Phe Ala Phe Met Ala Glu Lys Gly Lys Gin Ala 1 5 10 15

GAT TTC GTG TTG AGC GTT GGA GAT GAT AGA AGT GAT GAA GAC ATG TTT 95 Asp Phe Val Leu Ser Val Gly Asp Asp Arg Ser Asp Glu Asp Met Phe 20 25 30 GTG GCC ATT GGG GAT GGA ATA AAA AAG GGT CGG ATA ACT AAC AAC AAT 143

Val Ala He Gly Asp Gly He Lys Lys Gly Arg He Thr Asn Aεn Aεn 35 40 45

TCA GTG TTT ACA TGC GTA GTG GGA GAG AAA CCG AGT GCA GCT GAG TAC 191 Ser Val Phe Thr Cyε Val Val Gly Glu Lys Pro Ser Ala Ala Glu Tyr

50 55 60

TTT TTA GAC GAG ACG AAA GAT GTT TCA ATG ATG CTC GAG AAG CTC GGG 239 Phe Leu Asp Glu Thr Lys Asp Val Ser Met Met Leu Glu Lys Leu Gly 65 70 75

TGT CTC AGC AAC CAA GGA T GATGATCCGG AAGCTTCTCG TGATCTTTAT 288 Cys Leu Ser Asn Gin Gly 80 85

GAGTTAAAAG TTTTCGACTT TTTCTTCATC AAGATTCATG GGAAAGTTGT TCAATATGAA 348

CTTGTGTTTC TTGGTTCTGG ATTTTAGGGA GTCTATGGAT CC 390 (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 85 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: l ' 3

Ala Glu Lys He Phe Ala Phe Met Ala Glu Lys Gly Lys Gin Ala Asp 1 5 10 15

Phe Val Leu Ser Val Gly Asp Asp Arg Ser Asp Glu Asp Met Phe Val 20 25 30

Ala He Gly Asp Gly He Lys Lys Gly Arg He Thr Asn Asn Asn Ser 35 40 45 Val Phe Thr Cys Val Val Gly Glu Lys Pro Ser Ala Ala Glu Tyr Phe 50 55 60

Leu Asp Glu Thr Lys Asp Val Ser Met Met Leu Glu Lys Leu Gly Cys 65 70 75 80

Leu Ser Asn Gin Gly 85

(2) INFORMATION FOR SEQ ID NO: 30:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(in) HYPOTHETICAL: NO

(ni) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CCAIGGRTTI ACICKDATIG CICC 24

(2) INFORMATION FOR SEQ ID NO. 31:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(lil) HYPOTHETICAL: NO

(in) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

ATHGTIGTIW SIAAYMRIYT ICC 23 "&

( 2 ) INFORMATION FOR SEQ ID NO: 32:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(lli) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

YTITGGCCIA TITTYCAYTA 20

(2) INFORMATION FOR SEQ ID NO: 33.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(m) HYPOTHETICAL: NO

(in) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: TGRTCIARIA RYTCYTTIGC 20

(2) INFORMATION FOR SEQ ID NO. 34:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (m) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: TCRTCIGTRA ARTCRTCICC 20 I IS

(2) INFORMATION FOR SEQ ID NO: 35:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(ni) HYPOTHETICAL: NO

(ill) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

TTYGAYTAYG AYGGIACIYT 20

(2) INFORMATION FOR SEQ ID NO: 36:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(m) HYPOTHETICAL; NO

(ill) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GGIYTIWBNG CIGARCAYGG 20

(2) INFORMATION FOR SEQ ID NO: 37:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA

(in) HYPOTHETICAL. NO

(iii) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

ATIGCIAARC CIGTIATGAA 20 (2) INFORMATION FOR SEQ ID NO: 38:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(ill) HYPOTHETICAL: NO

(ill) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

CCIACIGTRC AIGCRAAIAC 20

(2) INFORMATION FOR SEQ ID NO: 39:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2982 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA to mRNA

(m) HYPOTHETICAL: NO

(m) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 64..2982 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

ATAAACTTCC TCGCGGCCGC CAGTGTGAGT AATTTAGTTT TGGTTCTGTT TTGGTGTGAG 60

CGT ATG CCT GGA AAT AAG TAC AAC TGC AGT TCT TCT CAT ATC CCA CTC 108 Met Pro Gly Asn Lys Tyr Asn Cys Ser Ser Ser His He Pro Leu 1 5 10 15

TCT CGA ACA GAA CGC CTC TTG AGA GAT AGA GAG CTT AGA GAG AAG AGG 156 Ser Arg Thr Glu Arg Leu Leu Arg Asp Arg Glu Leu Arg Glu Lys Arg 20 25 30

AAG AGC AAC CGA GCT CGT AAT CCT AAT GAC GTT GCT GGC AGT TCC GAG 204

Lys Ser Asn Arg Ala Arg Asn Pro Asn Asp Val Ala Gly Ser Ser Glu 35 40 45 i r 7

AAC TCT GAG AAT GAC TTG CGT TTA GAA GGT GAC AGT TCA AGG CAG TAT 252 Asn Ser Glu Asn Asp Leu Arg Leu Glu Gly Asp Ser Ser Arg Gin Tyr 50 55 60 GTT GAA CAG TAC TTG GAA GGG GCT GCT GCT GCA ATG GCG CAC GAT GAT 300 Val Glu Gin Tyr Leu Glu Gly Ala Ala Ala Ala Met Ala His Asp Asp 65 70 75

GCG TGT GAG AGG CAA GAA GTT AGG CCT TAT AAT AGG CAA CGA CTA CTT 348 Ala Cys Glu Arg Gin Glu Val Arg Pro Tyr Asn Arg Gin Arg Leu Leu 80 85 90 95

GTA GTG GCT AAC AGG CTC CCA GTT TCT CCC GTG AGA AGA GGT GAA GAT 396 Val Val Ala Asn Arg Leu Pro Val Ser Pro Val Arg Arg Gly Glu Asp 100 105 110

TCA TGG TCT CTT GAG ATC AGT GCT GGT GGT CTA GTC AGT GCT CTC TTA 444

Ser Trp Ser Leu Glu He Ser Ala Gly Gly Leu Val Ser Ala Leu Leu

115 120 125

GGT GTA AAG GAA TTT GAG GCC AGA TGG ATA GGA TGG GCT GGA GTT AAT 492

Gly Val Lys Glu Phe Glu Ala Arg Trp He Gly Trp Ala Gly Val Asn

130 135 140 GTG CCT GAT GAG GTT GGA CAG AAG GCA CTT AGC AAA GCT TTG GCT GAG 540 Val Pro Asp Glu Val Gly Gin Lys Ala Leu Ser Lys Ala Leu Ala Glu 145 150 155

AAG AGG TGT ATT CCC GTG TTC CTT GAT GAA GAG ATT GTT CAT CAG TAC 588 Lys Arg Cys He Pro Val Phe Leu Asp Glu Glu He Val His Gin Tyr 160 165 170 175

TAT AAT GGT TAC TGC AAC AAT ATT CTG TGG CCT CTG TTT CAC TAC CTT 636 Tyr Asn Gly Tyr Cys Asn Asn He Leu Trp Pro Leu Phe His Tyr Leu 180 185 190

GGA CTT CCG CAA GAA GAT CGG CTT GCC ACA ACC AGA AGC TTT CAG TCC 684 Gly Leu Pro Gin Glu Asp Arg Leu Ala Thr Thr Arg Ser Phe Gin Ser 195 200 205

CAA TTT GCT GCA TAC AAG AAG GCA AAC CAA ATG TTC GCT GAT GTT GTA 732 Gin Phe Ala Ala Tyr Lys Lys Ala Asn Gin Met Phe Ala Asp Val Val 210 215 220 AAT GAG CAC TAT GAA GAG GGA GAT GTC GTC TGG TGC CAT GAC TAT CAT 780 Asn Glu His Tyr Glu Glu Gly Asp Val Val Trp Cys His Asp Tyr His 225 230 235

CTT ATG TTC CTT CCT AAA TGC CTT AAG GAG TAC AAC AGT AAG ATG AAA 828 Leu Met Phe Leu Pro Lys Cys Leu Lys Glu Tyr Asn Ser Lys Met Lys 240 245 250 255

GTT GGA TGG TTT CTC CAT ACA CCA TTC CCT TCG TCT GAG ATA CAC AGG 876 Val Gly Trp Phe Leu His Thr Pro Phe Pro Ser Ser Glu He His Arg 260 265 270 I IQ

ACA CTT CCA TCA CGA TCA GAG CTC CTT CGG TCA GTT CTT GCT GCT GAT 924

Thr Leu Pro Ser Arg Ser Glu Leu Leu Arg Ser Val Leu Ala Ala Asp

275 280 285 TTA GTT GGC TTC CAT ACA TAT GAC TAT GCA AGG CAC TTT GTG AGT GCG 972

Leu Val Gly Phe His Thr Tyr Asp Tyr Ala Arg His Phe Val Ser Ala

290 295 300

TGC ACT CGT ATT CTT GGA CTT GAA GGA ACA CCT GAG GGA GTT GAG GAT 1020 Cys Thr Arg He Leu Gly Leu Glu Gly Thr Pro Glu Gly Val Glu Asp

305 310 315

CAA GGC AGG CTC ACT CGT GTA GCT GCT TTT CCA ATT GGC ATA GAT TCT 1068

Gin Gly Arg Leu Thr Arg Val Ala Ala Phe Pro He Gly He Asp Ser 320 325 330 335

GAT CGG TTT ATA CGA GCA CTT GAG GTC CCC GAA GTC AAA CAA CAC ATG 1116

Asp Arg Phe He Arg Ala Leu Glu Val Pro Glu Val Lys Gin His Met

340 345 350

AAG GAA TTG AAA GAA AGA TTT ACT GAC AGA AAG GTG ATG TTA GGT GTT 1164

Lyε Glu Leu Lys Glu Arg Phe Thr Asp Arg Lys Val Met Leu Gly Val

355 360 365 GAT CGT CTT GAC ATG ATC AAA GGG ATT CCA CAA AAG ATT CTG GCA TTC 1212

Asp Arg Leu Asp Met He Lys Gly He Pro Gin Lys He Leu Ala Phe

370 375 380

GAA AAA TTT CTC GAG GAA AAT GCA AAC TGG CGT GAT AAA GTG GTC TTA 1260 Glu Lys Phe Leu Glu Glu Asn Ala Asn Trp Arg Asp Lys Val Val Leu

385 390 395

TTG AAA ATT GCG GTG CCA ACA AGA CCT GAC GTT CCT GAG TAT CAA ACA 1308

Leu Lys He Ala Val Pro Thr Arg Pro Asp Val Pro Glu Tyr Gin Thr 400 405 410 415

CTC ACA AGC CAA GTT CAT GAA ATT GTT GGC CGC ATT ATT GGT CGT CTC 1356

Leu Thr Ser Gin Val His Glu He Val Gly Arg He He Gly Arg Leu

420 425 430

GGG ACA CTG ACT GCA GTT CCA ATA CAT CAT CTG GAT CGG TCT CTG GAC 1404

Gly Thr Leu Thr Ala Val Pro He His His Leu Asp Arg Ser Leu Asp

435 440 445 TTT CAT GCT TTA TGT GCA CTT TAT GCC GTC ACA GAT GTT GCG CTT GTA 1452

Phe His Ala Leu Cys Ala Leu Tyr Ala Val Thr Asp Val Ala Leu Val

450 455 460

ACA TCT TTG AGA GAT GGG ATG AAT CTT GTC AGT TAT GAG TTT GTT GCT 1500 Thr Ser Leu Arg Asp Gly Met Asn Leu Val Ser Tyr Glu Phe Val Ala

465 470 475

TGC CAA GAG GCC AAA AAG GGC GTC CTC ATT CTC AGT GAA TTT GCA GGT 1548

Cys Gin Glu Ala Lys Lys Gly Val Leu He Leu Ser Glu Phe Ala Gly 480 485 490 495 "°>

GCT GCA CAG TCT CTG GGT GCT GGA GCT ATT CTT GTG AAT CCT TGG AAC 1596 Ala Ala Gin Ser Leu Gly Ala Gly Ala He Leu Val Asn Pro Trp Asn 500 505 510 ATC ACA GAA GTT GCT GCC TCC ATT GGA CAA GCC CTA AAC ATG ACA GCT 1644 He Thr Glu Val Ala Ala Ser He Gly Gin Ala Leu Asn Met Thr Ala 515 520 525

GAA GAA AGA GAG AAA AGA CAT CGC CAT AAT TTT CAT CAT GTC AAA ACT 1692 Glu Glu Arg Glu Lys Arg His Arg His Asn Phe His His Val Lys Thr 530 535 540

CAC ACT GCT CAA GAA TGG GCT GAA ACT TTT GTC AGT GAA CTA AAT GAC 1740 His Thr Ala Gin Glu Trp Ala Glu Thr Phe Val Ser Glu Leu Asn Asp 545 550 555

ACT GTA ATT GAG GCG CAA CTA CGA ATT AGT AAA GTC CCA CCA GAG CTT 1788 Thr Val He Glu Ala Gin Leu Arg He Ser Lys Val Pro Pro Glu Leu 560 565 570 575

CCA CAG CAT GAT GCA ATT CAA CGG TAT TCA AAG TCC AAC AAC AGG CTT 1836 Pro Gin His Asp Ala He Gin Arg Tyr Ser Lys Ser Asn Asn Arg Leu 580 585 590 CTA ATC CTG GGT TTC AAT GCA ACA TTG ACT GAA CCA GTG GAT AAT CAA 1884 Leu He Leu Gly Phe Asn Ala Thr Leu Thr Glu Pro Val Aεp Asn Gin 595 600 605

GGG AGA AGA GGT GAT CAA ATA AAG GAG ATG GAT CTT AAT CTA CAC CCT 1932 Gly Arg Arg Gly Asp Gin He Lys Glu Met Asp Leu Asn Leu His Pro 610 615 620

GAG CTT AAA GGG CCC TTA AAG GCA TTA TGC AGT GAT CCA AGT ACA ACC 1980 Glu Leu Lys Gly Pro Leu Lys Ala Leu Cys Ser Asp Pro Ser Thr Thr 625 630 635

ATA GTT GTT CTG AGC GGA AGC AGC AGA AGT GTT TTG GAC AAA AAC TTT 2028

He Val Val Leu Ser Gly Ser Ser Arg Ser Val Leu Asp Lys Asn Phe

640 645 650 655

GGA GAG TAT GAC ATG TGG CTG GCA GCA GAA AAT GGG ATG TTC CTA AGG 2076

Gly Glu Tyr Asp Met Trp Leu Ala Ala Glu Asn Gly Met Phe Leu Arg 660 665 670 CTT ACG AAT GGA GAG TGG ATG ACT ACA ATG CCA GAA CAC TTG AAC ATG 2124 Leu Thr Asn Gly Glu Trp Met Thr Thr Met Pro Glu His Leu Asn Met 675 680 685

GAA TGG GTT GAT AGC GTA AAG CAT GTT TTC AAG TAC TTC ACT GAG AGA 2172 Glu Trp Val Asp Ser Val Lys His Val Phe Lys Tyr Phe Thr Glu Arg 690 695 700

ACT CCC AGG TCA CAC TTT GAA ACT CGC GAT ACT TCG CTT ATT TGG AAC 2220 Thr Pro Arg Ser His Phe Glu Thr Arg Asp Thr Ser Leu He Trp Asn 705 710 715 ' 2.0

TAC AAA TAT GCA GAT ATC GAA TTC GGG AGA CTT CAA GCA AGA GAT TTG 2268 Tyr Lys Tyr Ala Asp He Glu Phe Gly Arg Leu Gin Ala Arg Asp Leu 720 725 730 735 TTA CAA CAC TTA TGG ACA GGT CCA ATC TCT AAT GCA TCA GTT GAT GTT 2316 Leu Gin His Leu Trp Thr Gly Pro He Ser Asn Ala Ser Val Asp Val 740 745 750

GTC CAA GGA AGC CGC TCT GTG GAA GTC CGT GCA GTT GGT GTC ACA AAG 2364 Val Gin Gly Ser Arg Ser Val Glu Val Arg Ala Val Gly Val Thr Lys 755 760 765

GGA GCT GCA ATT GAT CGT ATT CTA GGA GAG ATA GTG CAT AGC AAG TCG 2412 Gly Ala Ala He Asp Arg He Leu Gly Glu He Val His Ser Lys Ser 770 775 780

ATG ACT ACA CCA ATC GAT TAC GTC TTG TGC ATT GGT CAT TTC TTG GGG 2460

Met Thr Thr Pro He Asp Tyr Val Leu Cys He Gly His Phe Leu Gly

785 790 795

AAG GAC GAA GAT GTT TAC ACT TTC TTC GAA CCA GAA CTT CCA TCC GAC 2508

Lys Asp Glu Asp Val Tyr Thr Phe Phe Glu Pro Glu Leu Pro Ser Asp

800 805 810 815 ATG CCA GCC ATT GCA CGA TCC AGA CCA TCA TCT GAC AGT GGA GCC AAG 2556 Met Pro Ala He Ala Arg Ser Arg Pro Ser Ser Asp Ser Gly Ala Lys 820 825 830

TCA TCA TCA GGA GAC CGA AGA CCA CCT TCA AAG TCG ACA CAT AAC AAC 2604 Ser Ser Ser Gly Asp Arg Arg Pro Pro Ser Lys Ser Thr His Asn Aεn 835 840 845

AAC AAA AGT GGA TCA AAA TCC TCA TCA TCC TCT AAC TCT AAC AAC AAC 2652 Asn Lys Ser Gly Ser Lys Ser Ser Ser Ser Ser Asn Ser Asn Asn Asn 850 855 860

AAC AAG TCC TCA CAG AGA TCT CTT CAG TCA GAG AGA AAA AGT GGA TCC 2700

Asn Lys Ser Ser Gin Arg Ser Leu Gin Ser Glu Arg Lys Ser Gly Ser

865 870 875

AAC CAT AGC TTA GGA AAC TCA AGA CGT CCT TCA CCA GAG AAG ATC TCA 2748

Asn His Ser Leu Gly Asn Ser Arg Arg Pro Ser Pro Glu Lys He Ser

880 885 890 895 TGG AAT GTG CTT GAC CTC AAA GGA GAG AAC TAC TTC TCT TGC GCT GTG 2796 Trp Asn Val Leu Asp Leu Lys Gly Glu Asn Tyr Phe Ser Cys Ala Val 900 905 910

GGT CGT ACT CGC ACC AAT GCT AGA TAT CTC CTT GGC TCA CCT GAC GAC 2844 Gly Arg Thr Arg Thr Asn Ala Arg Tyr Leu Leu Gly Ser Pro Asp Asp 915 920 925

GTC GTT TGC TTC CTT GAG AAG CTC GCT GAC ACC ACT TCC TCA CCT TAA 2892 Val Val Cys Phe Leu Glu Lys Leu Ala Asp Thr Thr Ser Ser Pro * 930 935 940 1 2. /

TAT CCC GAG ACA GTG TCA AGT GAG TTC ATG TAA CCC AAT AAA AAC TAT 2940

Tyr Pro Glu Thr Val Ser Ser Glu Phe Met * Pro Asn Lys Asn Tyr 945 950 955 TGT TTT GTA ACA AAA AGC AGC CAT TAC CAG ACT CTT TAG TGG 2982

Cys Phe Val Thr Lys Ser Ser His Tyr Gin Thr Leu * Trp 960 965 970

(2) INFORMATION FOR SEQ ID NO: 40:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 973 amino acids (D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

Met Pro Gly Asn Lys Tyr Asn Cys Ser Ser Ser His He Pro Leu Ser 1 5 10 15

Arg Thr Glu Arg Leu Leu Arg Asp Arg Glu Leu Arg Glu Lys Arg Lys 20 25 30

Ser Asn Arg Ala Arg Aεn Pro Asn Asp Val Ala Gly Ser Ser Glu Asn 35 40 45 Ser Glu Asn Asp Leu Arg Leu Glu Gly Asp Ser Ser Arg Gin Tyr Val 50 55 60

Glu Gin Tyr Leu Glu Gly Ala Ala Ala Ala Met Ala His Asp Asp Ala 65 70 75 80

Cys Glu Arg Gin Glu Val Arg Pro Tyr Asn Arg Gin Arg Leu Leu Val 85 90 95

Val Ala Asn Arg Leu Pro Val Ser Pro Val Arg Arg Gly Glu Asp Ser 100 105 110

Trp Ser Leu Glu He Ser Ala Gly Gly Leu Val Ser Ala Leu Leu Gly 115 120 125 Val Lys Glu Phe Glu Ala Arg Trp He Gly Trp Ala Gly Val Asn Val 130 135 140

Pro Asp Glu Val Gly Gin Lys Ala Leu Ser Lys Ala Leu Ala Glu Lys 145 150 155 160

Arg Cys He Pro Val Phe Leu Asp Glu Glu He Val His Gin Tyr Tyr 165 170 175

Asn Gly Tyr Cys Asn Asn He Leu Trp Pro Leu Phe His Tyr Leu Gly 180 185 190 Leu Pro Gin Glu Asp Arg Leu Ala Thr Thr Arg Ser Phe Gin Ser Gin 195 200 205

Phe Ala Ala Tyr Lys Lyε Ala Asn Gin Met Phe Ala Asp Val Val Asn 210 215 220

Glu His Tyr Glu Glu Gly Asp Val Val Trp Cys His Asp Tyr His Leu 225 230 235 240 Met Phe Leu Pro Lys Cys Leu Lys Glu Tyr Asn Ser Lys Met Lys Val

245 250 255

Gly Trp Phe Leu His Thr Pro Phe Pro Ser Ser Glu He His Arg Thr 260 265 270

Leu Pro Ser Arg Ser Glu Leu Leu Arg Ser Val Leu Ala Ala Asp Leu 275 280 285

Val Gly Phe His Thr Tyr Asp Tyr Ala Arg His Phe Val Ser Ala Cys 290 295 300

Thr Arg He Leu Gly Leu Glu Gly Thr Pro Glu Gly Val Glu Asp Gin 305 310 315 320 Gly Arg Leu Thr Arg Val Ala Ala Phe Pro He Gly He Asp Ser Asp

325 330 335

Arg Phe He Arg Ala Leu Glu Val Pro Glu Val Lys Gin His Met Lys 340 345 350

Glu Leu Lyε Glu Arg Phe Thr Asp Arg Lys Val Met Leu Gly Val Asp 355 360 365

Arg Leu Asp Met He Lys Gly He Pro Gin Lys He Leu Ala Phe Glu 370 375 380

Lys Phe Leu Glu Glu Asn Ala Asn Trp Arg Asp Lys Val Val Leu Leu 385 390 395 400 Lys He Ala Val Pro Thr Arg Pro Asp Val Pro Glu Tyr Gin Thr Leu

405 410 415

Thr Ser Gin Val His Glu He Val Gly Arg He He Gly Arg Leu Gly 420 425 430

Thr Leu Thr Ala Val Pro He His His Leu Asp Arg Ser Leu Asp Phe 435 440 445

His Ala Leu Cys Ala Leu Tyr Ala Val Thr Asp Val Ala Leu Val Thr 450 455 460

Ser Leu Arg Asp Gly Met Asn Leu Val Ser Tyr Glu Phe Val Ala Cys 465 470 475 480 Gin Glu Ala Lys Lys Gly Val Leu He Leu Ser Glu Phe Ala Gly Ala

485 490 495 /2,3

Ala Gin Ser Leu Gly Ala Gly Ala He Leu Val Asn Pro Trp Asn He 500 505 510

Thr Glu Val Ala Ala Ser He Gly Gin Ala Leu Asn Met Thr Ala Glu 515 520 525

Glu Arg Glu Lys Arg His Arg His Asn Phe His His Val Lys Thr His 530 535 540 Thr Ala Gin Glu Trp Ala Glu Thr Phe Val Ser Glu Leu Asn Asp Thr 545 550 555 560

Val He Glu Ala Gin Leu Arg He Ser Lys Val Pro Pro Glu Leu Pro 565 570 575

Gin His Asp Ala He Gin Arg Tyr Ser Lys Ser Asn Asn Arg Leu Leu 580 585 590

He Leu Gly Phe Asn Ala Thr Leu Thr Glu Pro Val Asp Asn Gin Gly 595 600 605

Arg Arg Gly Asp Gin He Lys Glu Met Asp Leu Asn Leu His Pro Glu 610 615 620 Leu Lys Gly Pro Leu Lys Ala Leu Cys Ser Asp Pro Ser Thr Thr He 625 630 635 640

Val Val Leu Ser Gly Ser Ser Arg Ser Val Leu Asp Lys Asn Phe Gly 645 650 655

Glu Tyr Asp Met Trp Leu Ala Ala Glu Asn Gly Met Phe Leu Arg Leu 660 665 670

Thr Asn Gly Glu Trp Met Thr Thr Met Pro Glu His Leu Asn Met Glu 675 680 685

Trp Val Asp Ser Val Lys His Val Phe Lys Tyr Phe Thr Glu Arg Thr 690 695 700 Pro Arg Ser His Phe Glu Thr Arg Asp Thr Ser Leu He Trp Asn Tyr 705 710 715 720

Lys Tyr Ala Asp He Glu Phe Gly Arg Leu Gin Ala Arg Asp Leu Leu 725 730 735

Gin His Leu Trp Thr Gly Pro He Ser Asn Ala Ser Val Asp Val Val 740 745 750

Gin Gly Ser Arg Ser Val Glu Val Arg Ala Val Gly Val Thr Lys Gly 755 760 765

Ala Ala He Asp Arg He Leu Gly Glu He Val His Ser Lys Ser Met 770 775 780 Thr Thr Pro He Asp Tyr Val Leu Cys He Gly His Phe Leu Gly Lys 785 790 795 800 Asp Glu Asp Val Tyr Thr Phe Phe Glu Pro Glu Leu Pro Ser Asp Met 805 810 815

Pro Ala He Ala Arg Ser Arg Pro Ser Ser Asp Ser Gly Ala Lys Ser 820 825 830

Ser Ser Gly Asp Arg Arg Pro Pro Ser Lys Ser Thr His Asn Asn Asn 835 840 845 Lys Ser Gly Ser Lys Ser Ser Ser Ser Ser Asn Ser Asn Asn Asn Asn 850 855 860

Lys Ser Ser Gin Arg Ser Leu Gin Ser Glu Arg Lys Ser Gly Ser Asn 865 870 875 880

His Ser Leu Gly Asn Ser Arg Arg Pro Ser Pro Glu Lys He Ser Trp 885 890 895

Asn Val Leu Asp Leu Lys Gly Glu Asn Tyr Phe Ser Cys Ala Val Gly 900 905 910

Arg Thr Arg Thr Asn Ala Arg Tyr Leu Leu Gly Ser Pro Asp Asp Val 915 920 925 Val Cys Phe Leu Glu Lys Leu Ala Asp Thr Thr Ser Ser Pro * Tyr 930 935 940

Pro Glu Thr Val Ser Ser Glu Phe Met * Pro Asn Lys Asn Tyr Cyε 945 950 955 960

Phe Val Thr Lys Ser Ser His Tyr Gin Thr Leu * Trp 965 970

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 300 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Oryza sativa

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: ATAAACTTCC TCGGACCAAA GAAGAGCATG TTGGTTGTGT CGGAGTTTAT TGGTTGCTCA 60 CCTTCACTGA GTGGAGCCAT TCGTGTTAAC CCGTGGAATA TCGAGGCAAC TGCAGAGGCA 120 CTGAATGAGG CCATCTCAAT GTCAGAGCGT AAAAGCAGCT GAGGCACGAA AAACATTACC 180

GTTATGTCAG CACCCATGAT GTTGCATATT GGTCTAAGAG CTTTGTACAG GACCTGGAGA 240 GGGCTTGCAA GGATCACTTT AGGAAACCAT GCTGGGGCAT TGGATTGGAT TTCGCTCAGG 300

(2) INFORMATION FOR SEQ ID NO: 42: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 627 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA to mRNA

(m) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Selagmella lepidophylla (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 4..627

(D) OTHER INFORMATION, /partial (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

ATT ATG TGG GTG CAT GAT TAC CAC CTC TGT CTG GTC CCT CAG ATG ATC 48

Met Trp Val His Asp Tyr His Leu Cys Leu Val Pro Gin Met He

1 5 10 15

CGC CAA AAG CTG CCA GAT GTG CAG ATT GGC TTC TTC CTC CAC ACC GCT 96

Arg Gin Lys Leu Pro Asp Val Gin He Gly Phe Phe Leu His Thr Ala

20 25 30 TTT CCC TCG TCA GAG GTC TTC CGC TGC TTG GCC GCA CGA AAG GAG CTG 144 Phe Pro Ser Ser Glu Val Phe Arg Cys Leu Ala Ala Arg Lys Glu Leu 35 40 45

CTG GAC GGC ATG CTT GGT GCC AAC TTG GTT GCT TTC CAG ACG CCA GAG 192 Leu Asp Gly Met Leu Gly Ala Asn Leu Val Ala Phe Gin Thr Pro Glu 50 55 60

TAT GCA CAC CAC TTC CTC CAG ACG TGC AGT CGC ATT TCT CTG CTG AAG 240 Tyr Ala His His Phe Leu Gin Thr Cys Ser Arg He Ser Leu Leu Lys 65 70 75

CAA CCG AGG AAG GCG TTC AGC TCG TTT CGT CAA TGT CTG GTC ATA ATG 288 Gin Pro Arg Lys Ala Phe Ser Ser Phe Arg Gin Cys Leu Val He Met 80 85 90 95 CAA GAA GCG CTA CGA GGG TCA AGA AGG TCA TCG TTG CGC GTG ACA AGC 336 Gin Glu Ala Leu Arg Gly Ser Arg Arg Ser Ser Leu Arg Val Thr Ser 100 105 110 TGA CAA CAT CGC GTG TAC GCG AGA AGC TTC TGT CGT ACG AGC TGT TCT 384 * Gin His Arg Val Tyr Ala Arg Ser Phe Cys Arg Thr Ser Cys Ser 115 120 125

TGA ACA AGA ACC CAC AGT GGA GGG ACA AGG TCG TTC TCA TTC AGG TTG 432 * Thr Arg Thr His Ser Gly Gly Thr Arg Ser Phe Ser Phe Arg Leu 130 135 140

CGA CCT CCA CGA CTG AGG ATT CTG AGC TTG CTG CGA CCG TAT CCG AAA 480 Arg Pro Pro Arg Leu Arg He Leu Ser Leu Leu Arg Pro Tyr Pro Lys 145 150 155

TTG TTA CAC GTA TTG ACG CTG TGC ACT CGA CGC TCA CAC ACA CCC ACT 528

Leu Leu His Val Leu Thr Leu Cys Thr Arg Arg Ser His Thr Pro Thr

160 165 170 175

CGT CTT CCT CAG GCA AGA CAT TGC GTT CTC GCA GTA CCT CGC ACT TCT 576

Arg Leu Pro Gin Ala Arg His Cys Val Leu Ala Val Pro Arg Thr Ser

180 185 190 CTC GAT CGC CGA TGC TCT TGC AAT CAA CTG TTC GAT GGC ATG AAC CTC 624

Leu Asp Arg Arg Cys Ser Cys Asn Gin Leu Phe Asp Gly Met Asn Leu 195 200 205

GTC 627 Val

(2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 208 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

Met Trp Val His Asp Tyr His Leu Cys Leu Val Pro Gin Met He Arg 1 5 10 15

Gin Lys Leu Pro Asp Val Gin He Gly Phe Phe Leu His Thr Ala Phe 20 25 30 Pro Ser Ser Glu Val Phe Arg Cys Leu Ala Ala Arg Lys Glu Leu Leu 35 40 45

Asp Gly Met Leu Gly Ala Asn Leu Val Ala Phe Gin Thr Pro Glu Tyr 50 55 60 Ala His His Phe Leu Gin Thr Cys Ser Arg He Ser Leu Leu Lys Gin 65 70 75 80

Pro Arg Lys Ala Phe Ser Ser Phe Arg Gin Cys Leu Val He Met Gin 85 90 95

Glu Ala Leu Arg Gly Ser Arg Arg Ser Ser Leu Arg Val Thr Ser * 100 105 110 Gin His Arg Val Tyr Ala Arg Ser Phe Cys Arg Thr Ser Cys Ser * 115 120 125

Thr Arg Thr His Ser Gly Gly Thr Arg Ser Phe Ser Phe Arg Leu Arg 130 135 140

Pro Pro Arg Leu Arg He Leu Ser Leu Leu Arg Pro Tyr Pro Lys Leu 145 150 155 160

Leu His Val Leu Thr Leu Cys Thr Arg Arg Ser His Thr Pro Thr Arg 165 170 175

Leu Pro Gin Ala Arg His Cys Val Leu Ala Val Pro Arg Thr Ser Leu

180 185 190 Asp Arg Arg Cys Ser Cys Asn Gin Leu Phe Asp Gly Met Asn Leu Val 195 200 205

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 645 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Selaginella lepidophylla

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

GGGTGGTTCT TGCACACGCC GTTTCCCTCG TCTGAGATTT ACAGAACGCT GCCGCTGCGG 60 GCCGAGCTGC TCCAAGGCGT CTTAGGCGCG GACTTAGTGG GGTTCCACAC ATACGACTAT 120

GCAAGGCACT TTGTTAGCGC GATGCACACG GATACTCGGG CTGGAAGGCA CTCCCAGGGT 180

GTCGAGGATC AAGGGAAGAT CACGCGAGTG GCTGCCTTCC CCGTGGATCG ATTCGGAGCG 240

ATTTATCGAC GCGTAGAGAC CGATGCGGTC AAGAAACACA TGCAAGAGCT GAGCCAGGTT 300 TTGCTGTCGT AAGGTTATGT TGGGGTGGAT AGGCTTGACA TGATTAAAGG AATTCCACAG 360

AAGCTGCTAG CCTTTGAAAA ATTCCTCGAG GAGAACTCCG AGTGGCGTGA TAAGGTCGTC 420 CTGGTGCAAA TCGCGGTGCC GACTAGAACG GACGTCCTCG AGTACCAAAA GCTTACGAGC 480

CAGGTTCACG AGATTGTTGG TCGCATAAAT GGACGTTTCG GCTCCTTGAC GGCTGTTCCT 540

ATCCATCACC TCGATCGGTC CATGAAATTT CCGGAGCTTT GTGCGTTATA TGCAATCACT 600

GATGTCCTGC TCGTGACATC CCTGCGCGAC GGCATGAACT TCGTC 645

(2) INFORMATION FOR SEQ ID NO: 45: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 498 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA to mRNA

(ill) HYPOTHETICAL. NO (ill) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

GCCGTTGTGG ATTCATCGCC TCGCACAAGC ACTCTTGTCG TGTCTGAGTT TATTGGATGC 60

TCACCTTCTT TGAGTGGTGC CATTAGGGTG AATCCATGGG ATGTGGATGC TGTTGCTGAA 120

GCGGTAAACT CGGCTCTTAA AATAGTGAGA CTGAGAAGCA ACTACGGCAT GAGAAACATT 180

ATCATTATAT TAGCACTCAT GATGTTGGTT ATTGGGCAAA GAGCTTTATG CAGGATCTTG 240 AGAGAGCGTG CCGAGATCAT TATAGTAAAC GTTGTTGGGG GATTGGTTTT GGCTTGGGGT 300

TCAGAGTTTT GTCACTCTCT CCAAGTTTTA GGAAGCTATC TGTGGACACA TTTGTTCCAG 360

TTTATAGGAA AACCACAGAG AGGGCTAATA TTCTTTTATA ATGGTACTCT TTGTTCCGAA 420

AGCTCATTGT TCAAGATCCA GCAACGGGTT CCTTGTCCTA AGCCCCTTAA GGCCCCATAA 480

CCGGTGTTTT TTAGTGAG ⁴⁹⁸ (2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 463 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA to mRNA

(m) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

GCCGTTGTGG ATTCATCGCC TCGCACAAGC ACTCTTGTCG TGTCTGAGTT TATTGGATGC 60

TCACCTTCTT TGAGTGGTGC CATTGGGTGA ATCCATGGGA TGTGGATGCT GTTGCTGAAG 120

CGGTAAACTC GGCTCTTAAA ATGAGTGAGA CTGAGAAGCA ACTACGGCAT GAGAAACATT 180

ATCATTATAT TAGCACTCAT GATGTTGGTT ATTGGGCAAA GAGCTTTATG CAGGATCTTG 240 AGAGAGCGTG CCGAGATCAT TATAGTAAAC GTTGTTGGGG GATTGGTTTT GGTTTGGGGT 300

TCAGAGTTTT TGTCACTCTC TCCAAGTTTA GGAAGCTATC TTGGGACAAT TGTTCCAGTT 360

TTTAGGGAAA ACACAGGGAA GGTTATTTCC TTGATTATAA TGGACCTTGT CCAAGCCCCA 420

TTTTTAAGGC CCAGGAACCG GGTTTTTTTT TCTTAAAGCC CCT 463

(2) INFORMATION FOR SEQ ID NO: 47- (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 394 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE- cDNA to mRNA

(m) HYPOTHETICAL: NO (ill) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

GGTATTGATG TAGAGGAAAT ACGTGGTGAA ATCGAAGAAA GCTGCAGGAG GATCAATGGA 60

GAGTTTGGGA AACCGGATAT CAACCTATCA TATATATTGA TACCCGGTTT CGATTAATGA 120

AATAAATGCT TATACCATAT TGCTGAGTGC GTGGTCGTTA CAGCTGTTAG AGATGGTATG 180

AACCTTACTC CCTACGAATA TATCGTTTGT AGACAAGGTT TACTTGGGTC TGAATCAGAC 240 TTTAGTGGCC CAAAGAAGAG CATGTTGGTT GCATCAAGTT TATTTGGATG TCCCCTTTCG 300 / 3o

CTTAGTGGGG CTATACGCGT AAACCCATGG AACCGTTGAA GCTACTTGAG GAGCCTTAAT 360 TAGGCCCCTC AAATATGCTG GAACACTACG GATG 394 (2) INFORMATION FOR SEQ ID NO: 48.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 428 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA to mRNA (m) HYPOTHETICAL: NO

(m) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Arabidopsis thaliana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

AAGTCCGTTG TGGATTCACG CCTCGCACAA GCACTCTTGT CGTGTCTAGT TTATTGGATG 60

CTCACCTTCT TTAGTGGTGC CATTAGGGTG AATCCATGGA TGTGGATGCT GTTGCTGAAG 120

CGGTAAACTC GGCTCTTAAA ATAGTGAGAC TGAGAAGCAA CTACGGCATG AGAAACATTA 180 TCATTATATT AGCACTCATG ATGTTGGTTA TTGGGCAAAG AGCTTTATGC AGGACTTAGA 240

GAGCGTGCCG AGATCATTAT AGTAAACGTT GTTGGGGGAT TGGTTTTGGT TTGGGGTTCA 300

AGTTTTGTCA CTCTCTCCAA GTTTTAGGAA GCTATCTTGT GGACACATTG TTCCAGTTTA 360

TAGAAACACA GGGAAGGGGC TATATTCTTG TTTAAATGGG ACCCCTTGTC CCTAAAAGTC 420

CCATTTGT 428 (2) INFORMATION FOR SEQ ID NO: 49:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 481 base pairs

(B) TYPE: nucleic acid (C! STRANDEDNESS: double

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO

(ill) ANTI-SENSE: NO

(Vl) ORIGINAL SOURCE: (A) ORGANISM: Arabidopsis thaliana ' 3 /

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

CAAACGAAGA GCTTCGTGGG AAAGTGGTTC TCGTGCAGAT TACTAATCCT GCTCGTAGTT 60 CAGGTAAGGA TGTTCAAGAT GTAGAGAAAC AGATAAATTT ATTGCTGATG AGATCAATTC 120

TAAATTTGGG AGACCTGGTG GTTATAAGCC TATTGTTTTG TAATGGACCT GTTAGTACTT 180

TGGATAAAGT TGCTTATTAC GCGATCTCGG AGTGTGTTGT CGTGAATCTG TGAGAGATGG 240

GATGAATTTG GTGCCTTATA AGTACACAGT GACTCGGCAA GGGAGCCCTG CTTTGGATGC 300

AGCTTTGGTT TTGGGGAGGA TGATGTTAGG AAGAGTGTGA TTATTGTTTC TGAGGTTCAA 360 CCGGTTGTCC TCCATCTCTA GTGGTGCGAT CCCTTTTAAT CCGTGGACAT CGATCAGCAC 420

TTACGCCATG AGCTTCAAAT CCGGTTTCCG CAAAGGGAAA ATTGCCCCGA GCTTAAGGCC 480

A 481

(2) INFORMATION FOR SEQ ID NO: 50:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 395 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA to mRNA

(ill) HYPOTHETICAL: NO

(m) ANTI-SENSE: NO (vi) ORIGINAL SOURCE.

(A) ORGANISM: Arabidopsis thaliana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: AGACCTGGTG GTTATAAGCC TATTGTGTTT GTCAATGGAC CTGTTAGTAC TTTGGATAAA 60

TTGCTTATTA CGCGATCTCG GAGTGTGTTG TCGTGAATCT GTGAGAGATG GGATGAATTT 120

GGTGCCTTAT AAGTACACAG TGACTCGGCA AGGGAGCCCT GCTTTGGATG CAGCTTTAGG 180

TTTTGGGGAG GATGATGTTA GGAAGAGTGT GATTATTGTT TCTAGTTCAT CGGTTGTCTC 240

CATCTCTGAG TGGTGCGATC CGTTAATCCG TGGAACATCG TGCAGTCACT AAACGCCATG 300 AGCCTGCAAT ACGATGTCGC AAAGGGAAAA TCTTTGCCAC CAGAAGCATC ATAAGTACAT 360

AAAGCCTCAC AATTGCCTAT TTGGGCCGGG GTTTT 395 / 3 2.

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 431 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Oryza sativa

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 1

(D) OTHER INFORMATION: /standard_name= "GENBANK ID: D22143"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

GGGAATGGAG GGTCTCCGAG CTGCAGCAGC AATTTGAGGG GAAGACTGTG TTGCTCGGTG 60

TGGATGACAT GGATATCTTC AAGGGTATCA ACTTGAAGCT TCTTGCCTTC GAGAATATGT 120 TGAGGACACA TCCCAAGTGG CAGGGGCGGG CAGTGTTGGT GCAAATTGCT AATCCGGCCC 180

GTGGAAAGGG TAAGGATCTT GAAGCCATCC AGGCTGAGAT TCATGAGAGC TGCAAGAGGA 240

TTAATGGAGA GTTTGGCCAG TCAGGATACA GCCCTGTTGT CTTCATTGAC CGTGATGTGT 300

CAAGTGTGGA GGAAGATTGC CTACTACACA ATAGCAGAAT GTGTGGTGGT GACTGCTGTT 360

AGGGATGGGA TTGACTTGAC ACCATATGGA TATATTGTCT GTAGGGCAGG GGTCTTACTC 420 ACATCAGAGG T 431

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 496 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO / 33

(vi) ORIGINAL SOURCE.

(A) ORGANISM Oryza sativa

(ix) FEATURE: (A) NAME/KEY- mιsc_feature

(B) LOCATION- 1

(D) OTHER INFORMATION, /standard_name= "GENBANK ID D40048

(xi) SEQUENCE DESCRIPTION SEQ ID NO- 52

CTACCGTTCC CTCCCTGTTC GCGACGAGAT CCTCAAATCA CTGCTAAACT GCGATCTGAT 60

TGGGTTCCAC ACCTTTGATT ACGCGCGGCA TTTCCTGTCC TGCTGCAGCC GGATGCTGGG 120 GATCGAGTAC CAGTCGAAGA GGGGATATAT CGGTCTCGAT TACTTTGGCC GCACTGTTGG 180

GATAAAGATC ATGCCTGTTG GGATTAACAT GACGCAGCTG CAGACGCAGA TCCGGCTGCC 240

TGATCTTGAG TGGCGTGTCG CGAACTCCGG AAGCAGTTTG ATGGGAAGAC TGTCATGCTC 300

GGTGTGGATG ATATGGACAT ATTTAAGGGG ATTAATCTGA AAGTTCTTGC GTTTTGAGCA 360

GATGCTGAGG ACACACCCAA AATGGCAGCC AAGGCAGTTT TGGTGCAGAT TCAAACCAAG 420 GGTGGTTGTT GGGAGGACTT AGGTACAGCT AGATATGAGT TCAGGGGTAA TGACATTTCA 480

GGCGGTATTT CCTTGG 496

(2) INFORMATION FOR SEQ ID NO. 53:

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 288 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- double (D) TOPOLOGY, linear

(ll) MOLECULE TYPE cDNA to mRNA (m) HYPOTHETICAL: NO (ill) ANTI-SENSE NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM. Oryza sativa

(xi) SEQUENCE DESCRIPTION. SEQ ID NO 53

GGACCAAAGA AGAGCATGTT GGTTGTGTCG GAGTTTATTG GTTGCTCACC TTCACTGAGT 60 GGAGCCATTC GTGTTAACCC GTGGAATATC GAGGCAACTG CAGAGGCACT GAATGAGGCC 120

ATCTCAATGT CAGAGCGTAA AAGCAGCTGA GGCACGAAAA ACATTACCGT TATGTCAGCA 180

CCCATGATGT TGCATATTGG TCTAAGAGCT TTGTACAGGA CCTGGAGAGG GCTTGCAAGG 240

ATCACTTTAG GAAACCATGC TGGGGCATTG GATTGGATTT CGCTCAGG 288 (2) INFORMATION FOR SEQ ID NO: 54:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2207 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (ill) HYPOTHETICAL: NO (m) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM- Solanum tuberosum

(B) STRAIN: Kardal

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 161..1906

(ix) FEATURE:

(A) NAME/KEY: mιsc_feature (B) LOCATION: 842..850

(D) OTHER INFORMATION: /functιon= "putative glycosylatronsite"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

CTTTTCTGAG TAATAACATA GGCATTGATT TTTTTTCAAT TAATAACACC TGCAAACATT 60

CCCATTGCCG GCATTCTCTG TTCTTACAAA AAAAAACATT TTTTTGTTCA CATAAATTAG 120 TTATGGCATC AGTATTGAAC CCTTTAACTT GTTATACAAT ATG GGT AAA GCT ATA 175

Met Gly Lys Ala He

1 5

ATT TTT ATG ATT TTT ACT ATG TCT ATG AAT ATG ATT AAA GCT GAA ACT 223 He Phe Met He Phe Thr Met Ser Met Asn Met He Lys Ala Glu Thr

10 15 20

TGC AAA TCC ATT GAT AAG GGT CCT GTA ATC CCA ACA ACC CCT TTA GTG 271

Cys Lys Ser He Asp Lys Gly Pro Val He Pro Thr Thr Pro Leu Val 25 30 35

ATT TTT CTT GAA AAA GTT CAA GAA GCT GCT CTT CAA ACT TAT GGC CAT 319

He Phe Leu Glu Lys Val Gin Glu Ala Ala Leu Gin Thr Tyr Gly His 40 45 50

AAA GGG TTT GAT GCT AAA CTG TTT GTT GAT ATG TCA CTG AGA GAG AGT 367 Lys Gly Phe Asp Ala Lys Leu Phe Val Asp Met Ser Leu Arg Glu Ser 55 60 65 ' 35

CTT TCA GAA ACA GTT GAA GCT TTT AAT AAG CTT CCA AGA GTT GTG AAT 415

Leu Ser Glu Thr Val Glu Ala Phe Asn Lys Leu Pro Arg Val Val Asn

70 75 80 85 GGT TCA ATA TCA AAA AGT GAT TTG GAT GGT TTT ATA GGT AGT TAC TTG 463

Gly Ser He Ser Lys Ser Asp Leu Asp Gly Phe He Gly Ser Tyr Leu

90 95 100

AGT AGT CCT GAT AAG GAT TTG GTT TAT GTT GAG CCT ATG GAT TTT GTG 511 Ser Ser Pro Asp Lys Asp Leu Val Tyr Val Glu Pro Met Asp Phe Val

105 110 115

GCT GAG CCT GAA GGC TTT TTG CCA AAG GTG AAG AAT TCT GAG GTG AGG 559

Ala Glu Pro Glu Gly Phe Leu Pro Lys Val Lys Asn Ser Glu Val Arg 120 125 130

GCA TGG GCA TTG GAG GTG CAT TCA CTT TGG AAG AAT TTA AGT AGG AAA 607

Ala Trp Ala Leu Glu Val His Ser Leu Trp Lys Asn Leu Ser Arg Lys

135 140 145

GTG GCT GAT CAT GTA TTG GAA AAA CCA GAG TTG TAT ACT TTG CTT CCA 655

Val Ala Asp His Val Leu Glu Lys Pro Glu Leu Tyr Thr Leu Leu Pro

150 155 160 165 TTG AAA AAT CCA GTT ATT ATA CCG GGA TCG CGT TTT AAG GAG GTT TAT 703

Leu Lys Asn Pro Val He He Pro Gly Ser Arg Phe Lys Glu Val Tyr

170 175 180

TAT TGG GAT TCT TAT TGG GTA ATA AGG GGT TTG TTA GCA AGC AAA ATG 751 Tyr Trp Asp Ser Tyr Trp Val He Arg Gly Leu Leu Ala Ser Lys Met

185 190 195

TAT GAA ACT GCA AAA GGG ATT GTG ACT AAT CTG GTT TCT CTG ATA GAT 799

Tyr Glu Thr Ala Lys Gly He Val Thr Asn Leu Val Ser Leu He Asp 200 205 210

CAA TTT GGT TAT GTT CTT AAC GGT GCA AGA GCA TAC TAC AGT AAC AGA 847

Gin Phe Gly Tyr Val Leu Asn Gly Ala Arg Ala Tyr Tyr Ser Asn Arg

215 220 225

AGT CAG CCT CCT GTC CTG GCC ACG ATG ATT GTT GAC ATA TTC AAT CAG 895

Ser Gin Pro Pro Val Leu Ala Thr Met He Val Asp He Phe Asn Gin

230 235 240 245 ACA GGT GAT TTA AAT TTG GTT AGA AGA TCC CTT CCT GCT TTG CTC AAG 943

Thr Gly Asp Leu Asn Leu Val Arg Arg Ser Leu Pro Ala Leu Leu Lys

250 255 260

GAG AAT CAT TTT TGG AAT TCA GGA ATA CAT AAG GTG ACT ATT CAA GAT 991 Glu Asn His Phe Trp Asn Ser Gly He His Lys Val Thr He Gin Asp

265 270 275

GCT CAG GGA TCA AAC CAC AGC TTG AGT CGG TAC TAT GCT ATG TGG AAT 1039

Ala Gin Gly Ser Asn His Ser Leu Ser Arg Tyr Tyr Ala Met Trp Asn 280 285 290 ; 3 <ff

AAG CCC CGT CCA GAA TCG TCA ACT ATA GAC AGT GAA ACA GCT TCC GTA 1087

Lys Pro Arg Pro Glu Ser Ser Thr He Asp Ser Glu Thr Ala Ser Val 295 300 305 CTC CCA AAT ATA TGT GAA AAA AGA GAA TTA TAC CGT GAA CTG GCA TCA 1135

Leu Pro Asn He Cys Glu Lys Arg Glu Leu Tyr Arg Glu Leu Ala Ser

310 315 320 325

GCT GCT GAA AGT GGA TGG GAT TTC AGT TCA AGA TGG ATG AGC AAC GGA 1183 Ala Ala Glu Ser Gly Trp Asp Phe Ser Ser Arg Trp Met Ser Asn Gly

330 335 340

TCT GAT CTG ACA ACA ACT AGT ACA ACA TCA ATT CTA CCA GTT GAT TTG 1231

Ser Asp Leu Thr Thr Thr Ser Thr Thr Ser He Leu Pro Val Asp Leu 345 350 355

AAT GCA TTC CTT CTG AAG ATG GAA CTT GAC ATT GCC TTT CTA GCA AAT 1279

Asn Ala Phe Leu Leu Lys Met Glu Leu Asp He Ala Phe Leu Ala Asn 360 365 370

CTT GTT GGA GAA AGT AGC ACG GCT TCA CAT TTT ACA GAA GCT GCT CAA 1327

Leu Val Gly Glu Ser Ser Thr Ala Ser His Phe Thr Glu Ala Ala Gin 375 380 385 AAT AGA CAG AAG GCT ATA AAC TGT ATC TTT TGG AAC GCA GAG ATG GGG 1375

Asn Arg Gin Lys Ala He Asn Cys He Phe Trp Asn Ala Glu Met Gly

390 395 400 405

CAA TGG CTT GAT TAC TGG CTT ACC AAC AGC GAC ACA TCT GAG GAT ATT 1423 Gin Trp Leu Aεp Tyr Trp Leu Thr Asn Ser Asp Thr Ser Glu Asp He

410 415 420

TAT AAA TGG GAA GAT TTG CAC CAG AAC AAG AAG TCA TTT GCC TCT AAT 1471

Tyr Lys Trp Glu Asp Leu His Gin Asn Lys Lys Ser Phe Ala Ser Asn 425 430 435

TTT GTT CCG CTG TGG ACT GAA ATT TCT TGT TCA GAT AAT AAT ATC ACA 1519

Phe Val Pro Leu Trp Thr Glu He Ser Cys Ser Asp Asn Aεn He Thr 440 445 450

ACT CAG AAA GTA GTT CAA AGT CTC ATG AGC TCG GGC TTG CTT CAG CCT 1567

Thr Gin Lys Val Val Gin Ser Leu Met Ser Ser Gly Leu Leu Gin Pro 455 460 465 GCA GGG ATT GCA ATG ACC TTG TCT AAT ACT GGA CAG CAA TGG GAT TTT 1615

Ala Gly He Ala Met Thr Leu Ser Asn Thr Gly Gin Gin Trp Asp Phe

470 475 480 485

CCG AAT GGT TGG CCC CCC CTT CAA CAC ATA ATC ATT GAA GGT CTC TTA 1663 Pro Asn Gly Trp Pro Pro Leu Gin His He He He Glu Gly Leu Leu

490 495 500

AGG TCT GGA CTA GAA GAG GCA AGA ACC TTA GCA AAA GAC ATT GCT ATT 1711

Arg Ser Gly Leu Glu Glu Ala Arg Thr Leu Ala Lys Asp He Ala He 505 510 515 ,3j

CGC TGG TTA AGA ACT AAC TAT GTG ACT TAC AAG AAA ACC GGT GCT ATG 1759 Arg Trp Leu Arg Thr Asn Tyr Val Thr Tyr Lys Lys Thr Gly Ala Met 520 525 530 TAT GAA AAA TAT GAT GTC ACA AAA TGT GGA GCA TAT GGA GGT GGT GGT 1807 Tyr Glu Lys Tyr Asp Val Thr Lys Cys Gly Ala Tyr Gly Gly Gly Gly 535 540 545

GAA TAT ATG TCC CAA ACG GGT TTC GGA TGG TCA AAT GGC GTT GTA CTG 1855 Glu Tyr Met Ser Gin Thr Gly Phe Gly Trp Ser Asn Gly Val Val Leu 550 555 560 565

GCA CTT CTA GAG GAA TTT GGA TGG CCT GAA GAT TTG AAG ATT GAT TGC 1903 Ala Leu Leu Glu Glu Phe Gly Trp Pro Glu Asp Leu Lys He Asp Cys 570 575 580

TAATGAGCAA GTAGAAAAGC CAAATGAAAC ATCATTGAGT TTTATTTTCT TCTTTTGTTA 1963

AAATAAGCTG CAATGGTTTG CTGATAGTTT ATGTTTTGTA TTACTATTTC ATAAGGTTTT 2023

TGTACCATAT CAAGTGATAT TACCATGAAC TATGTCGTTC GGACTCTTCA AATCGGATTT 2083

TGCAAAAATA ATGCAGTTTT GGAGAATCCG ATAACATAGA CCATGTATGG ATCTAAATTG 2143 TAAACAGCTT ACTATATTAA GTAAAAGAAA GATGATTCCT CTGCTTTAAA AAAAAAAAAA 2203

AAAA 2207

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 581 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

Met Gly Lys Ala He He Phe Met He Phe Thr Met Ser Met Asn Met

1 5 10 15

He Lys Ala Glu Thr Cys Lys Ser He Asp Lys Gly Pro Val He Pro 20 25 30

Thr Thr Pro Leu Val He Phe Leu Glu Lys Val Gin Glu Ala Ala Leu

35 40 45 Gin Thr Tyr Gly His Lys Gly Phe Asp Ala Lys Leu Phe Val Asp Met

50 55 60

Ser Leu Arg Glu Ser Leu Ser Glu Thr Val Glu Ala Phe Asn Lys Leu 65 70 75 80 / 3Θ

Pro Arg Val Val Asn Gly Sei He Ser Lys Ser Asp Leu Asp Gly Phe 85 90 95

He Gly Ser Tyr Leu Ser Ser Pro Asp Lys Asp Leu Val Tyr Val Glu 100 105 110

Pro Met Asp Phe Val Ala Glu Pro Glu Gly Phe Leu Pro Lys Val Lys 115 120 125 Asn Ser Glu Val Arg Ala Trp Ala Leu Glu Val Hiε Ser Leu Trp Lys 130 135 140

Asn Leu Ser Arg Lys Val Ala Asp His Val Leu Glu Lys Pro Glu Leu 145 150 155 160

Tyr Thr Leu Leu Pro Leu Lys Asn Pro Val He He Pro Gly Ser Arg 165 170 175

Phe Lys Glu Val Tyr Tyr Trp Asp Ser Tyr Trp Val He Arg Gly Leu 180 185 190

Leu Ala Ser Lys Met Tyr Glu Thr Ala Lys Gly He Val Thr Asn Leu 195 200 205 Val Ser Leu He Asp Gin Phe Gly Tyr Val Leu Asn Gly Ala Arg Ala 210 215 220

Tyr Tyr Ser Asn Arg Ser Gin Pro Pro Val Leu Ala Thr Met He Val 225 230 235 240

Asp He Phe Asn Gin Thr Gly Asp Leu Aεn Leu Val Arg Arg Ser Leu 245 250 255

Pro Ala Leu Leu Lys Glu Asn Hiε Phe Trp Asn Ser Gly He His Lys 260 265 270

Val Thr He Gin Asp Ala Gin Gly Ser Asn His Ser Leu Ser Arg Tyr 275 280 285 Tyr Ala Met Trp Asn Lys Pro Arg Pro Glu Ser Ser Thr He Asp Ser 290 295 300

Glu Thr Ala Ser Val Leu Pro Asn He Cys Glu Lys Arg Glu Leu Tyr 305 310 315 320

Arg Glu Leu Ala Ser Ala Ala Glu Ser Gly Trp Asp Phe Ser Ser Arg 325 330 335

Trp Met Ser Asn Gly Ser Asp Leu Thr Thr Thr Ser Thr Thr Ser He 340 345 350

Leu Pro Val Asp Leu Asn Ala Phe Leu Leu Lys Met Glu Leu Asp He 355 360 365 Ala Phe Leu Ala Asn Leu Val Gly Glu Ser Ser Thr Ala Ser His Phe 370 375 380 Thr Glu Ala Ala Gin Asn Arg Gin Lys Ala He Asn Cys He Phe Trp 385 390 395 400

Asn Ala Glu Met Gly Gin Trp Leu Asp Tyr Trp Leu Thr Aεn Ser Asp 405 410 415

Thr Ser Glu Asp He Tyr Lys Trp Glu Asp Leu His Gin Asn Lys Lys 420 425 430 Ser Phe Ala Ser Asn Phe Val Pro Leu Trp Thr Glu He Ser Cys Ser 435 440 445

Asp Asn Asn He Thr Thr Gin Lys Val Val Gin Ser Leu Met Ser Ser 450 455 460

Gly Leu Leu Gin Pro Ala Gly He Ala Met Thr Leu Ser Asn Thr Gly 465 470 475 480

Gin Gin Trp Asp Phe Pro Asn Gly Trp Pro Pro Leu Gin His He He 485 490 495

He Glu Gly Leu Leu Arg Ser Gly Leu Glu Glu Ala Arg Thr Leu Ala 500 505 510 Lys Asp He Ala He Arg Trp Leu Arg Thr Asn Tyr Val Thr Tyr Lys 515 520 525

Lys Thr Gly Ala Met Tyr Glu Lys Tyr Asp Val Thr Lys Cys Gly Ala 530 535 540

Tyr Gly Gly Gly Gly Glu Tyr Met Ser Gin Thr Gly Phe Gly Trp Ser 545 550 555 560

Asn Gly Val Val Leu Ala Leu Leu Glu Glu Phe Gly Trp Pro Glu Asp 565 570 575

Leu Lys He Asp Cys 580 (2) INFORMATION FOR SEQ ID NO: 56

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: CTCAGATCTG GCCACAAA 18 (2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTGCTCGTCT GCAGGTGC 18

Claims

' 4- 1

C LAIMS

1. Method of modification of the development and/or composition of cells, tissue or organs in vivo by inducing a change in the metabolic availability of trehalose-6-phosphate.

2. Method for the stimulation of carbon flow in the glycolytic direction in a cell by decreasing the intracellular availability trehalose-6-phosphate.

3. Method for the inhibition of carbon flow in the glycolytic direction in a cell by increasing the intracellular availability of trehalose-6-phosphate.

4. Method for the inhibition of photosynthesis in a cell by decreasing the intracellular availability of trehalose-6-phosphate.

5. Method for the stimulation of photosynthesis in a cell by increasing the intracellular availability of trehalose-6-phosphate.

6. Method for the stimulation of sink-related activity by increasing the intracellular availability of trehalose-6-phosphate.

7. Method for the stimulation of growth of a cell or tissue by decreasing the intracellular availability of trehalose-6-phosphate.

8. Method for the inhibition of growth of a cell or tissue by increasing the intracellular availability of trehalose-6-phosphate.

9. Method for increasing metabolism of cells by decreasing the intracellular availability of trehalose-6-phosphate.

10. Method according to claim 2, 4, 7 or 9, characterized in that said decrease of the intracellular concentration of trehalose-6- phosphate is effected by an increase in trehalose-phosphate- phosphatase (TPP) activity. l ^jή- 2-

11 Method according claim 10, characterized in that the increase in TPP activity is achieved by transformation of said cells with a vector capable of expression of the enzyme TPP

12 Method according to claim 11, characterized in that said cells are transformed with a vector comprising a heterologous gene encoding TPP

13 Method according to claim 2, 4, 7 or 9, characterized in that said decrease of the intracellular concentration of trehalose-6- phosphate is effected by a decrease in trehalose-phosphate synthase (TPS) activity.

14 Method according to claim 13, characterized in that said decrease in TPS activity is effected by transformation of said cells with a vector capable of expresεion of a molecule that inhibits TPS

15 Method according to claim 14, characterized in that said vector comprises the antisense gene of TPS

16 Method according to claim 10, characterized in that said decrease is due to mutation of the endogenous TPP enzyme

17 Method according to claim 10, characterized in that the decrease of trehalose-6-phosphate is effected by the relative overexpression of a phospho-alpha- (1, 1) -glucosidase

18 Method according to claim 3, 5, 6 or 8, characterized in that said increase of the intracellular concentration of trehalose-6- phosphate is effected by an increase in TPS activity

19 Method according claim 18 characterized in that the increase in TPS activity is achieved by transformation of said cells with a vector capable of expression of the enzyme TPS

20 Method according to claim 19, characterized in that said cells are transformed with a vector comprising a heterologous gene encoding TPS ' 4"3

21 Method according to claim 3, 5, 6 or 8, characterized in that said increase of the intracellular concentration of trehalose-6- phosphate is effected by a decrease in TPP activity.

22 Method according to claim 21, characterized in that said decrease in TPP activity is effected by transformation of said cells with a vector capable of expression of a molecule that inhibits TPP.

23. Method according to claim 22, characterized in that said vector comprises the antisense gene of TPS.

24. Method according to claim 18, characterized m that said increase is due to a mutation of the endogenous TPS enzyme.

25. Method according to any one of claims 1-24, characterized in that said cell or cells are located in a plant.

26. Method according to claim 25, characterized in that said plant is a transgenic plant

27. Method according to claim 26, characterized m that said transgenic plant is produced by transformation with Agrobacterium tumefaciens .

28. Method according to any one of claims 1-24, characterized in that said cell or cells are located in an animal, preferably a mammal, more preferably a human being.

29. Method according to any one of claims 1-24, characterized in that said cells are microorganisms, preferably a microorganism selected from the group consisting of bacteria, microbes, yeasts, fungi, cell cultures, oocytes, sperm cells, hybridomas, Protista and callus.

30. A cloning vector which comprises a gene coding for TPP. ' 4-4-

31. The cloning vector of claim 30, characterized in that it comprises a nucleotide sequence selected from the group of nucleotide sequences depicted in SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:17 and the parts coding for TPP from the bipartite enzymes as coded by SEQ ID NO: 24, SEQ ID NO- 28, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO. 44.

32. A cloning vector which comprises an antisense gene for TPS, which upon expression is able to prevent functional activity of the endogenous TPS gene

33. A cloning vector which comprises a gene for TPS, characterized in that it comprises a nucleotide sequence selected from the group of nucleotide sequences depicted in SEQ ID NO 1, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,

SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 39, SEQ ID NO:

41, SEQ ID NO: 42, SEQ ID NO- 44, SEQ ID NO- 45, SEQ ID NO: 46, SEQ ID

NO: 47, SEQ ID NO: 48, SEQ ID NO. 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO. 52 and SEQ ID NO: 53.

34. A cloning vector which comprises an antisense gene for TPP, which upon expression is able to prevent functional activity of the endogenous TPP gene

35. Plant characterized in that it or one of its ancestors is transformed with a vector comprising the nucleotide sequence coding for TPP.

36. Plant characterized in that it or one of its ancestors is transformed with a vector comprising the nucleotide sequence coding for an antisense gene of TPP.

37. Plant characterized in that it or one of its ancestors is transformed with a vector comprising the nucleotide sequence coding for an antisense gene of TPS.

38. Use of trehalose-6-phosphate to influence carbohydrate partitioning in cells. l 4-5 39 Use of trehalose-6-phosρhate to increase biomasε.

40. Use of trehalose-6-phosphate to affect hexokinase activity.

41. Use of trehalose-6-phosphate to affect hexokinase signalling function.

42. Use of trehalose-6-phosphate to affect cell wall synthesis.

43. Use of compounds influencing the intracellular availability of trehalose-6-phosphate to increase biomass.

44. Use of compounds influencing the intracellular availability of trehalose-6-phosphate to affect hexokinase activity

45. Use of compounds influencing the intracellular availability of trehalose-6-phosphate to affect photosynthesis.

46. Use of compounds influencing the intracellular availability of trehalose-6-phosphate to affect the carbon flow m the glycolytic pathway.

47 Method for the prevention of cold sweetening by increasing the intracellular availability of trehalose-6-phosphate.

48. Method for the inhibition of mvertaεe in beet after harveεt by increasing the intracellular availability of trehalose-6-phosρhate.

49. Use of compounds influencing the intracellular availability of trehalose-6-phosphate to affect cold sweetening or invertase inhibition.

50. Method according to claim 47 or 48, characterized in that increasing the intracellular availability of T-6-P results from the increase of trehalose phosphate synthase activity. 51 Method according to claim 47, characterized in that the regulation of the availability of T-6-P is specifically altered in potato tubers .

52. Method according to claim 51, characterized m that a gene coding for trehalose phosphate synthase is specifically expressed in tubers

53. Method according to claim 52, characterized in that said gene is the TPS gene from Escherichi a coli .

54. Method according to claim 48, characterized in that the regulation of the availability of T-6-P is specifically altered in beet taproots .

55. Method according to claim 54, characterized in that a gene coding for trehalose phosphate synthase is specifically expressed in taproots .

56. Method for the accumulation of trehalose, characterized in that an organism is transformed with a DNA sequence coding for a bipartite TPS-TPP enzyme.

57. Method according to claim 56, characterized m that said gene is the bipartite gene from Arabidopsiε thaliana

58. Method according to claim 56, characterized in that said gene is the bipartite gene from Selagmel la l epidophylla .

59. Method according to claim 56, characterized in that said gene is the human bipartite gene

60. Method according to claim 56, characterized in that said gene is the bipartite gene from Helianthus annuus .

61. Method to prevent metabolic steering during the production of trehalose by expression of a DNA sequence coding for a bipartite TPS- TPP enzyme. "τ-7

62 Method according to claims 1-24, characterized in that expression of TPP or TPS is limited to a specific tissue

63 Method according to claims 1-24, characterized in that expression of TPP or TPS is under control of an inducible promoter

64 Method of modification of the development and/or composition of cells, tissue or organs m vivo by inducing a change m the metabolic availability of trehalase.

65 Method for the stimulation of carbon flow in the glycolytic direction in a cell by increasing the intracellular availability trehalase

66 Method for the inhibition of carbon flow in the glycolytic direction in a cell by decreasing the intracellular availability of trehalase.

67 Method for the stimulation of sink-related activity by decreasing the intracellular availability of trehalase

68. Method for the stimulation of growth of a cell or tissue by increasing the intracellular availability of trehalase

69 Method for the inhibition of growth of a cell or tissue by decreasing the intracellular availability of trehalase

70. Method for increasing metabolism of cells by increasing the intracellular availability of trehalase.

71. Method for the stimulation of carbon flow in the glycolytic direction m a cell by expression of trehalose-6-phosphate phosphatase.

72. Method for the inhibition of carbon flow m the glycolytic direction in a cell by expression of trehalose-6-phosphate εynthase. I -fc5>

73. Method for the inhibition of photosynthesis in a cell by expression of trehalose-6-phosphate phosphatase.

74. Method for the stimulation of photosynthesis in a cell by expression of trehalose-6-phosphate synthase.

75. Method for the stimulation of sink-related activity by expression of trehalose-6-phosphate synthase.

76. Method for the stimulation of growth of a cell or tissue by expression of trehalose-6-phosphate phosphatase.

77. Method for the inhibition of growth of a cell or tissue by expression of trehalose-6-phosphate synthase.

78. Method for increasing metabolism of cells by expression of trehalose-6-phosphate phosphatase

79. Method for the prevention of cold sweetening by expression of trehalose-6-phosphate synthase.

80. Method for the prevention of cold sweetening by decreasing the availability of intracellular trehalase.

81. Method for the prevention of bolting by decreasing the intracellular availability of trehalose-6-phosphate.

82. Method for the prevention of bolting by expression of trehalose-6-phosphate phosphatase.

83. Method for the induction of bolting by increasing the intracellular availability of trehalose-6-phosphate

84. Method for the induction of bolting by expression of trehalose- 6-phosphate synthase.

85. Method for the induction of bolting by decreasing the intracellular availability of trehalase. 86 Method for increasing the yield of plants by transforming them with an enzyme coding for trehalose-6-phosphate phosphatase

87. Method for increasing the yield of plants by increasing the intracellular availability of trehalose-6-phosphate

88. Polynucleotide coding for trehalose-6-phosphate synthase, characterized in that it is a bipartite enzyme which has a mutation in the part coding for trehalose-6-phosphate phosphatase

89. Polynucleotide coding for trehalose-6-phosphate phosphatase, characterized in that it is a bipartite enzyme which has a mutation in the part coding for trehalose-6-phosphate synthase.

90 Polynucleotide according to claim 88 or 89, characterized in that the bipartite gene is human TPS/TPP

91 Polynucleotide according to claim 90, characterized in that the human TPS/TPP has an amino acid sequence according to SEQ ID NO. 11

92 Polynucleotide according to claim 91, characterized in that it comprises the polynucleotide sequence of SEQ ID NO:10

93 Polynucleotide according to claim 88 or 89, characterized in that the bipartite gene is Arabidopsis thal iana TPS/TPP

94 Polynucleotide according to claim 93, characterized in that the human TPS/TPP has an ammo acid sequence according to SEQ ID NO: 40.

95. Polynucleotide according to claim 94, characterized in that it comprises the polynucleotide sequence of SEQ ID NO:39

96 Polynucleotide according to claim 88 or 89, characterized in that the bipartite gene is Selagmella lepi dophylla TPS/TPP

97 Polynucleotide according to claim 96, characterized in that the human TPS/TPP comprises an ammo acid sequence according to SEQ ID NO. 43 or a mutein thereof 98. Polynucleotide according to claim 97, characterized m that it comprises the polynucleotide sequence of SEQ ID NO:42 or SEQ ID NO:44.

99. Polynucleotide according to claim 88 or 89, characterized in that the bipartite gene is Helianthus annuus TPS/TPP

100. Polynucleotide according to claim 99, characterized in that the human TPS/TPP comprises an ammo acid sequence according to SEQ ID NO. 25 or a mutein thereof.

101. Polynucleotide according to claim 100, characterized in that it comprises the polynucleotide sequence of SEQ ID NO:24 or SEQ ID NO:26 or SEQ ID NO:28.

102. Vector harbouring a polynucleotide accordmg to any of claims 88 to 101.

103 Host organism comprising a vector according to claim 102.

104 Host organism according to claim 103, characterized m that it is Agrobacterium tumefaciens .

105. Cell transformed with a host organism according to claim 103 or 104.

106 Cell according to claim 105, characterized in that it is a plant cell.

107. Plant or plant part, regenerated from the plant cell according to claim 106.